Funded under USAID Cooperative Agreement No. 663-A-00-00-0358-00.

Produced in collaboration with the Ethiopia Public Health Training Initiative, The Carter

Center, the Ethiopia Ministry of Health, and the Ethiopia Ministry of Education.

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©2006 by Abilo Tadesse, Meseret Alem

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PREFACE

Text book on Medical Bacteriology for Medical Laboratory Technology students are not available as need, so this lecture note will alleviate the acute shortage of text books and reference materials on medical bacteriology.

Since it comprises most of the contents of course outline on medical bacteriology to nursing, pharmacy and environmental science students, it can be used as a main learning material to these category of students.

This lecture note gives emphasis on the knowledge and procedures of medical bacteriology to common pathogens in our country.

At last but not least, the quality of this lecture note is kept updated by continous comments made by users of this lecture note.

Abilo Tadesse

Meseret Alem

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ACKNOWLEDGEMENT

We would like to acknowledge the Carter Center, USA, for financial support for the preparation of this lecture note.

Our deepest gratitude goes to Prof. Dennis Carlson for his invaluable technical and moral support for the completion of this work.

We also extend our appreciation to those individuals who reviewed this lecture note in different teaching institutions for the materialization of this lecture note.

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	TABLE OF CONTENTS
		Page
Preface ………………………………………………………………...i
Acknowledgement ……………………………………………………ii
Table of Contents		iii
List of tables	..............................................................................	vii
List of figures	.............................................................................	viii
List of Abbreviation …………………………………………………..xi
CHAPTER ONE
1.1.	Introduction to Microbiology	1
1.2.	The Microbial World	5
1.3.	Structure of bacteria	12
1.4.	Classification of bacteria	23
1.5.	Cultivation of bacteria	33
1.6.	Bacterial nutrition ………………………	47
1.7.	Bacterial growth	49
1.8.	Bacterial genetics	85
1.9.	Sterilization and disinfection	92
1.10. Antimicrobial sensitivity testing		107
CHAPTER TWO
Collection, transport, and examination of specimen		113

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CHAPTER THREE
	3.1 Gram positive cocci		173
	3.1.1. Genus Staphylococci		173
	3.1.2. Genus Streptococci		180
	3.2 Gram positive spore forming rods		192
	3.2.1. Genus Bacillus ……………………		192
	3.2.2.	Genus Clostridium	197
	3.2.3. Gram positive Non-spore forming rods		205
	3.3.1. Genus Corynebacteria		205
	3.3.2. Genus Listeria		210
	3.3.3. Genus Erysipelothrix		212
3.4	Gram negative diplococci		213
	3.4.1	Genus Neisseria	213
3.5	Gram negative coccobacilli		221
	3.5.1 Genus Haemophilus		221
	3.5.2 Genus Bordetella		224
	3.5.3 Genus Brucella		227
	3.5.4 Genus Francissella		229
	3.5.5 Genus Pasteurella		230
3.6 Gram negative rods			231
	3.6.1 Genus Escherichia		233
	3.6.2. Genus Klebsiella		235
	3.6.3. Genus Enterobacter		236
	3.6.4. Genus Citrobacter		237
	3.6.5. Genus Salmonella		237
	3.6.6. Genus Shigella		242
	3.6.7. Genus Proteus		244
	3.6.8. Genus Yersinia		245

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3.6.9. Genus Peudomonas		249
3.6.10 Genus Vibrios		252
3.6.11	Genus Campylobacter	254
3.6.12	Genus Helicobacter	256
3.7. Genus Mycobacteria		263
3.8. Spirochetes		273
3.8.1.Genus Treponema		273
3.8.2 Genus Borellia		279
3.8.3 Genus Leptospira		281
3.9 Genus Rickettsia		282
3.10. Genus Mycoplasma		286
3.11. Genus Chlamydia		288
CHAPTER FOUR
4.1. Host-parasite relationship		294
4.2. Normal microbial flora		300
4.3. Infection of skin and wound		304
4.4. Infection of respiratory tract		307
4.5. Infection of gastrointestinal tract		313
4.6. Infection of urinary tract		318
4.7. Infection of genital tract		321
4.8. Infection of blood		325
4.9. Infection of central nervous system		327
4.10 Infection of bone and joint		331
CHAPTER FIVE
5.1. Bacteriology of water		335
	v

CHAPTER SIX
Food Bacteriology	341
Annexes	375
Glossary	429
References	433

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LIST OF TABLES
Table1.1 The distinguishing features between eukaryotic and
prokaryotic cell	11
Table 1.2 Comparison between flagella and pili	22
Table 2.1 Differentiation of staphylococcal species	180
Table 2.2 Classification based on hemolytic reaction of
Streptococci	183
Table 2.3 Comparison of streptolysin	185
Table 2.4 Differentiation of streptococci species	192
Table 2.5 Comparison features of meningococcal meningitis and
meningococcemia	219
Table 2.6 Comparison features of N.gonorrhea and N.
Meningitides	221
Table 2.7 Comparison between tuberculoid and lepromatous
Leprosy	267
Table 2.8 Hosts and vectors of medically impotant rickettsiae 283
Table 4.1 Characteristic of bacterial toxin	295
Table 4.2 Examples of food intoxication	317
Table 4.3 Examples of food infection	317
Table 4.4 Causative agents and disease of genital infection ..	321
Table 4.5 Genital ulcer with or with out inguinal
Lymphadenopathy	324
Table 4.6 Cerebrospinal fluid findings in meningitis	330

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LIST OF FIGURES
Fig 1.1Ultrastructure of bacteria	13
Fig 1.2 Cell wall of Gram positive and Gram negative bacteria .14
Fig 1.3 Components of Bacterial flagellum	20
Fig 1.4 Flagellar arrangement	21
Fig 1.5 Morphology bacteria	24
Fig 1.6 Inoculation technique	43
Fig 1.7 Inoculation of solid culture media in petridishes	44
Fig 1.8 Inoculation of slant and butt media	45
Fig 1.9 Inoculation of slant media	46
Fig 1.10 Co2-enriched atmosphere	47
Fig 1.11 Bacterial growth curve	51
Fig 1.12 Bacterial chromosome	86
Fig 1.13 Gene transfer by Transformation	89
Fig 1.14 Gene transfer by Transduction	90
Fig 1.15 Gene transfer by conjugation	91
Fig 1.16 Antimicrobial sensitivity test media	111
Fig 3.1 Staphylococci	175
Fig 3.2 Streptococci	182
Fig 3.3 Streptococcus pneumoniae	189
Fig 3.4 Neisseria gonorrhea	214
Fig 3.5 Neisseria meningitides	218
Fig 2.6 Spirochetes	273

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ABBEREVIATIONS

. AIDS……………… Acquired immunodeficiency syndrome

. AFB ……………… Acid fast bacilli

. ATP ……………… Adenosine triphosphate

. CO2………………. Carbon dioxide

. CSF……………… Cerebrospinal fluid

. CNS ……………… Central nervous system

. DNA ……………… Deoxy ribonucleotide

. DNase …………… Deoxy ribonucleotidase

. GIT ………………. Gastrointestinal tract

. HIV ………………. Human immunodeficiency virus

. HPF ……………… High power field

. IP ………………….. Incubation period

. LGV ………………. Lymphogranuloma venereum

. NADase ………….. Nicotinamide adenine dinucleotidase

. NB ………………… Notta Bonne

. OC ………………… Degree of Celsius

. PH …………………. Hydrogen ion concentration

. RBC ………………. Red blood cell

. RNA ………………. Ribonucleotide

. RPR ……………….. Rapid plasma reagin

. SS agar …………… Salmonella-Shigella agar

. STD ……………….. Sexually transmitted disease

. UTI ………………… Urinary tract infection

. VDRL ………… Venereal disease research laboratory test

. WBC ………………. White blood cell

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CHAPTER ONE

Learning Objective

•At the end of the lesson, the student should be able to:

1.Identify the structure of bacterial cell

2.Do simple and differential staining methods

3.Describe the essential nutrients required for bacterial growth

4.Describe the mechanisms of genetic variation in bacterial cell

5.Identify the chemical meanses of sterilization and disinfection, and their effect on bacterial cell

6.Do and interpret the result of anti-microbial sensitivity testing in vitro

1.1 INTRODUCTION TO MICROBIOLOGY

Microbiology is a subject which deals with living organisms that are individually too small to be seen with the naked eye.

It considers the microscopic forms of life and deals about their reproduction, physiology, and participation in the process of nature, helpful and harmful relationship with other living things, and significance in science and industry.

Subdivision of microbiology

Bacteriology deals about bacteria.

Mycology deals about fungi.

Virology deals about viruses.

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History of Microbiology

Man kind has always been affected by diseases which were originally believed to be visitations by the gods and meant to punish evil doers.

Hippocratus, father of medicine, observed that ill health resulted due to changes in air, winds, water, climate, food, nature of soil and habits of people.

Varro (117-26 BC)said a theory that disease was caused by animated particles invisible to naked eye but which were carried in the air through the mouth and nose into the body.

Fracastorius (1500 G.C.) proposed that the agents of communicable disease were living germs, that could be transmitted by direct contact with humans and animals, and indirectly by objects ; but no proof because of lacking experimental evidence.

Antony Van Leeuwenhoek (1632-1723 G.C.), father of Microbiology, observed “animalcules” using simple microscope with one lens.

He was the first who properly described the different shapes of bacteria.

Although Leeuwenhoek was not concerned about the origin of micro-organism; many other scientists were searching for an explanation for spontaneous appearance of living things from decaying meat, stagnating ponds, fermenting grains and infected wounds.

On the bases of this observation, two major theories were formulated.

1.Theory of Abiogenesis

2.Theory of Biogenesis

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Theory of Abiogenesis deals with the theory of spontaneous generation; stating that living things originated from non-living things. Aristotle (384-322 BC): The founder of a theory spontaneous generation.

He observed spontaneous existence of fishes from dried ponds, when the pond was filled with rain.

Francesco Redi (1626-1697): He is the scientist who first tried to set an experiment to disprove spontaneous generation.

-He put the meat in a bottle and covered it with a gauze.

-He observed that the flies laid eggs from which the maggots developed.

-He said maggots did not developed from meat but from flies egg.

Theory of Biogenesis states that life comes from pre-existing life. Louis Pasteur (1822-1895 GC) was the scientist who disproved the theory of abiogenesis.

He designed a large curved flask (Pasteur goose neck flask) and placed a sterile growth broth medium. Air freely moved through the tube; but dust particles were trapped in the curved portion of flask.

Microbial growth in the broth was not seen.

Therefore Pasteur proved that micro-organisms entered to substrates through the air and micro-organisms did not evolve spontaneously.

Major contribution of Louis Pasteur

1.Microbial theory of fermentation

2.Principles and practice of sterilization and pasteurization

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3.Control of diseases of silk worms

4.Development of vaccines against anthrax and rabies.

5.Discovery of streptococci

The germ theory of disease

The complete establishment of the germ theory of disease depended on the work of a German scientist, Robert Koch (1843- 1910).

Major achievements of Robert Koch

1.Discovery and use of solid medium in bacteriology

2.Discovery of causative agents of tuberculosis and cholera.

3.Koch’s phenomenon

4.Koch’s postulates

Koch’s postulates: proof of germ theory of disease

A micro-organism can be accepted as a causative agent of an infectious disease only if the following conditions are satisfied.

1.The micro-organism should be found in every case of the disease and under conditions which explain the pathological changes and clinical features.

2.It should be possible to isolate the causative agent in pure culture from the lesion.

3.When such pure culture is inoculated into appropriate laboratory animal, the lesion of the disease should be reproduced.

4.It should be possible to reisolate the bacterium in pure culture from the lesion produced in the experimental animal.

5.Now a days additional postulate is mentioned i.e.

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Specific antibody to the bacterium should be detectable in the serum during the course of the disease.

It has not been possible to fulfil every one of Koch’s postulates, but by adhering to them as closely as possible, serious errors have been prevented.

Exceptions to Koch’s postulates

1.Many healthy people carry pathogens but do not exhibit symptoms of the disease.

2.Some microbes are very difficult or impossible to grow in vitro(in the laboratory) in artificial media. Eg. Treponema pallidum

3.Many species are species specific. Eg. Brucella abortus cause abortion in animals but no report in humans.

4.Certain diseases develop only when an opportunistic pathogen invades immunocompromised host.

1.2. THE MICROBIAL WORLD

TAXONOMIC CLASSIFICATION OF ORGANISMS

TAXONOMY is the science of organisimal classification. Classification is the assignment of organisms (species) into anorganised scheme of naming .idealy these schemes are based on evolutionary relationships (i.e the more similar the name, the closer the evolutionary relationships). Thus, classification is concerned with:-

1.The establishment of criteria for identifying organisms & assignment to groups (what belongs where)

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2.The arrangement of organisms into groups of organism of organism (e.g. At what level of diversity should a single species be split in to two or more species?).

3.Consideration of how evolution resulted in the formation these groups.

TAXON:-

„A group or category of related organisms. Two key characteristics of taxa are:

-Members of lower level taxa (e.g. Species) are more similar to each other than are members of higher level taxa (eg.Kingdom or domain).

-Member of specific taxa are more similar to each other than any are to members of different specific taxa found at the same hierarchical level (eg. Humans are more similar to apes, i.e., comparison between species, than either is similar to, for example, Escherichia coli). Thus once you know that two individuals are member of the same taxon, you can inter certain similarities between the two organisms.

NOTE that taxa are dynamic, changing as our knowledge of organism and evolutionary relationships change

BINOMIAL NOMENCLATURE

-Organisms are named using binomial nomenclature ( viruses are exceptions)

-Binomial nomenclature employs the names of the two level taxa, genus and species, to name a specie. Binomial nomenclature includes:

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i.Genus comes before species (e.g., Escherichia coli)

ii.Genus name is always capitalized (e.g., Escherichia)

iii.Species name is never capitalized (e.g., coli)

iv.Both names are always either italicized or underlined ( e.g Escherichia coli )

v.The genus name may be used alone, but not the species name (i.e saying or writing “Escherichia “ alone is legitimate while saying or writing “ coli” is not)

Strain

a)A strain in some ways is equivalent to a breed or subspecies among plants or animal. Strain is the level below the species

b)Two members of the same strain are more similar to each other than either is to an individual that is a member of a different strain, even if all three organisms are members of the same species

Bacterial species

-A bacterial species is defined by the similarities found among its members. Properties such as biochemical reactions, chemical composition, cellular structures, genetic characteristics, and immunological features are used in defining a bacterial species. Identifying a species and determining its limits presents the most challenging aspects of biological classification for any type of organism.

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-A formal means of distinguishing bacterial species is by employing a dichotomous key to guide the selection of test used to efficiently determine those bacterial properties most relevant to bacterial identification

The five kingdom system

The five kingdom system was first proposed in 1969 and is showing its age

The five kingdoms include:

i.Plantae ( the plants)

ii.Fungi ( the fungi)

iii.Animalia ( the animals )

iv.Protista ( the unicellular eukaryotes)

v.Monera ( the prokaryotes)

Kingdom of Monera

Three categories:

-Eubacteria

Are our common, everyday bacteria, some of which are disease – causing; also the taxon from which mitochondria originated.

-Cyanobacteria

Are photosynthetic eubacteria, the taxon from which chloroplast originated

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-Archaeobacteria

Are distinctive in their adaptation to extreme environments (e.g., very hot, salty, or acidic) though not all archaeobacteria live in extreme environments.

These distinctions are more phenotypic than they are evolutionary (i.e., a cyanobacteria is a eubacteria, and neither is an archaebacteria).

Kingdom Protista

Protista like Monera consist mostly of unicellular organisms. Distinctively, however, the members of Kingdom Protista are all eukaryotic while the mebers of kingdom Monera are all prokaryotic. Some members of protista are multicellular, however Kingdom protista represents a grab bag, essentially the place where the species are classified when they are not classified as either fungi, animals or plants.

Kingdom Fungi

Unlike pprotists, the eukaryotic fungi are typically non – aquatic species. They traditionally are nutrients absorbers plus have additional distinctive features. They do exist unicellular fungi, which we call yeast

DOMAIN

The domain is a taxanomic category that, depending on point of view, is either above the level of kingdom or supercedes the kingdom. The domain system contains three members

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¾Eukaryotes ( domain Eukarya )

¾Eubacteria ( domain Bacteria)

¾Archaebacteria ( domain Archaea)

Viral classification

Classification of viruses is not nearly as well developed as the classification of cellular organisms. Today viruses tend to be classified by their chemical, morphological and physiological attributes (e.g. genome = DNA vs RNA, virion particle = enveloped vs non enveloped and myriad details of their intracellular infection cycles). Binomial nomenclature is not employed to name viruses; instead viruses are named by their common names (e.g., Human Immunodeficiency Virus a.k.a HIV)

Dichotomous key

A means of assigning an organism to a specific taxonomic category typically involves the use of specific criteria that may posed as questions ( e.g. What does the organism look like etc. ). Relevant criteria may be arranged as a dichotomous key. In a dichotomous key questions are arranges hierarchically with more general questions are asked first, with questions becoming more specific asked subsequently

EUKARYOTIC CELL

Eu- true

Karyote- nucleus

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The eukaryotic cell has a true membrane bound nucleus, usually containing multiple chromosomes, a mitotic apparatus, a well defined endoplasmic reticulum and mitochondria.

PROKARYOTIC CELL

Pro- primitive Karyote- nucleus

The prokaryotic cell possesses naked DNA with out associated basic proteins, divides amitotically by binary fission and bounded by a semi rigid cell wall.

Table 1.1. The distinguishing features between

Eukaryotic cell and Prokaryotic cell

Features	Prokaryotic cell	Eukaryotic cell
.Size	1μm	10μm
. Nuclear membrane	Absent	Present
. Chromosome	Single	Multiple
. Nucleolus	Absent	Present
. Histones	Absent	Present
. Sexual reproduction	Absent	Present
. Cytoplasmic ribosomes	70s	80s
. Mitochondria	Absent	Present
. Endoplasmic reticulum	Absent	Present
. Lysosomes	Absent	Present.
	11

. Micro filaments and tubules Absent		Present
. Site of oxidativre
phosphorylation	Cell membrane	Mitochondria
. Site of photosynthesis	Cell membrane	Chloroplast
. Peptidoglycan	Present	Absent
. Cell membrane
composition	Phospholipids & Proteins	Sterols

Bacterial Cell

General property:

•Typical prokaryotic cell

•Contain both DNA and RNA

•Most grow in artificial media

•Replicate by binary fission

•Almost all cotain rigid cell wall

•Sensitive to antimicrobial agent

1.3.STRUCTURE OF BACTERIA

Bacterial structure is considered at three levels.

1.Cell envelope proper: Cell wall and cell membrane.

2.Cellular element enclosed with in the cell envelope: Mesosomes, ribosomes, nuclear apparatus, polyamies and cytoplasmic granules.

3.Cellular element external to the cell envelope: Flagellum, Pilus and Glycocalyx.

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Fig. 1.1 Ultrastructure of Bacteria

1.Cell envelope proper A. Cell wall

Multi layered structure and constitutes about 20% of the bacterial dry weight.

Average thickness is 0.15-0.5 μm.

Young and rapidly growing bacteria has thin cell wall but old and slowly dividing bacteria has thick cell wall.

It is composed of N-acetyl Muramic acid and N-acetyl Glucosamine back bones cross linked with peptide chain and pentaglycine bridge.

Components of cell wall of Gram negative bacteria

1.Peptidoglycan

2.Lipoprotein

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3.Phospholipid

4.Lipopolysaccharide

Components of cell wall of Gram positive bacteria

1.Peptidoglycan

2.Teichoic acid

Fig. 1.2 Cell wall of Gram Positive & Gram Negative Bacteria

Functions of cell wall

1.Provides shape to the bacterium

2.Gives rigidity to the organism

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3.Protects from environment

4.Provides staining characteristics to the bacterium

5.Contains receptor sites for phages/complements

6.Site of action of antibody and colicin

7.Contains toxic components to host

Bacteria with defective cell walls

Bacteria with out cell wall can be induced by growth in the presence of antibiotics and a hypertonic environment to prevent lysis.

They are of three types:

1.Protoplasts: Derived from Gram-positive bacteria and totally lacking cell walls; unstable and osmotically fragile; produced artificially by lysozyme and hypertonic medium: require hypertonic conditions for maintenance.

2.Spheroplast: Derived from Gram-negative bacteria; retain some residual but non-functional cellwall material; osmotically fragile;produced by growth with penicillin and must be maintained in hypertonic medium.

3.L- forms: Cell wall-deficient forms of bacteria usually produced in the laboratory but sometimes spontaneously formed in the body of patients treated with penicillin; more stable than protoplasts or spheroplasts , they can replicate in ordinary media.

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B. Cell membrane

Also named as cell membrane or cytoplasmic membrane It is a delicate trilaminar unit membrane .

It accounts for 30% of the dry weight of bacterial cell.

It is composed of 60% protein, 20-30% lipids and 10-20% carbohydrate.

Function of cell membrane

1.Regulates the transport of nutrients and waste products into and out of the cell.

2.Synthesis of cell wall components

3.Assists DNA replication

4.Secrets proteins

5.Carries on electron transport system

6.Captures energy in the form of ATP

2. Cellular element enclosed with in the cell envelope

A. Mesosomes

Convoluted invagination of cytoplasmic membrane often at sites of septum formation.

It is involved in DNA segregation during cell division and respiratory enzyme activity.

B. Ribosomes

Cytoplasmic particles which are the sites of protein synthesis.

It is composed of RNA(70%) and proteins(30%) and constitutes 90% of the RNA and 40% of the total protein.

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The ribosome monomer is 70s with two subunits, 30s and 50s.

C. Polyamines

They are of three types

. Putrescin

. Spermidine

.Spermine

It is found in association with bacterial DNA, ribosomes and cell membrane.

Function of polyamines

1.Antimutagenic.

2.Prevent dissociation of 70s ribosome into subunits.

3.Increase resistance of protoplast lysis.

D. Cytoplasmic granules

. represent accumulated food reserves.

Nature of granules

. Glycogen

. Poly-beta hydroxy butyrate

. Babes-ernst (Volutin)

E. Nuclear apparatus

Well defined nucleus and nuclear membrane , discrete chromosome and mitotic apparatus are not present in bacteria ; so nuclear region of bacteria is named as nuclear body, nuclear apparatus and nucleoid.

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Bacterial genome consists of single molecule of double stranded DNA arranged in a circular form.

Besides nuclear apparatus, bacteria may have extra chromosomal genetic material named as plasmids.

Plasmids do not play any role in the normal function of the bacterial cell but may confer certain additional properties(Eg. Virulence, drug resistance) which may facilitate survival and propagation of the micro- organism.

3. Cellular element external to the cell envelope

A.Glycocalyx (capsule and slime layer) Capsule is gel firmly adherent to cell envelope. Slime is gel easily washed off from cell envelope. All bacteria have at least a thin slime layer.

Capsule is composed of polysaccharide and protein(D-Glutamate of Bacillus anthracis)

Features of capsule

1.Usually weakly antigenic.

2.Not necessary for viability.

3.Endows virulence.

4.Protects from phagocytosis.

5.Capsulated strains are invariably non-motile.

6.Visualized by negative staining and capsule staining.

7.Detected by quellung phenomenon.

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B. Flagellum

It is the organ of locomotion in bacterial cell and consists of thee parts. These are .The filament

. The hook

. The basal body

The basal body and hook are embedded in the cell surface while the filament is free on the surface of bacterial cell.

Their presence in bacterial cell is detected by

. Hanging drop preparation

. Swarming phenomenon on surface of plate agar

. Motility media

. Special staining methods

. Silver impregnation methods

. Dark –field microscopy

. Electron microscopy

Size: 3-20μm in length and 0.01-0.013μm in diameter. It is composed of protein named as flagellin.

The flagellar antigen in motile bacterium is named as H (Hauch) antigen.

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Fig. 1.3 Components of Bacterial Flagellum

Flagellar arrangements

1.Atrichous: Bacteria with no flagellum.

2.Monotrichous: Bacteria with single polar flagellum.

3.Lophotrichous: Bacteria with bunch of flagella at one pole.

4.Amphitrichous: Bacteria with flagella at both poles.

5.Peritrichous: Bacteria with flagella all over their surface.

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Fig. 1.4 Flagellar arrangements

Endoflagella (axial filament)

It is the organ of motility found in periplasmic space of spirochetes.

C. Pili (fimbriae)

It is hair like structure composed of protein (pilin)

Two types (Based on function)

. Common pili: The structure for adherence to cell surface.

. Sex pili: The structure for transfer of genetic material from the donor to the recipient during the process of conjugation.

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. Spherical central

2.Bulging of cell wall

. Oval sub terminal

. Oval terminal

. Spherical terminal

. Free spore

1.4. Classification of bacteria

Bacterial classification depends on the following characteristics.

1.Morphology and arrangement

2.Staining

3.Cultural characteristics

4.Biochemical reactions

5.Antigenic structure

6.Base composition of bacterial DNA

Morphology and staining of bacteria are the commonly used characteristics to classify bacteria.

1. Morphology of bacteria

When bacteria are visualized under light microscope, the following morphology are seen.

1.Cocci (singular coccus): Round or oval bacteria measuring about 0.5-1.0μmb in diameter.They are found insingle, pairs, chains or clusters.

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2.Bacilli (singular bacillus): Stick-like bacteria with rounded, tepered, square or swollen ends; with a size measuring 1-10μm in length by 0.3-1.0μm in width.

3.Coccobacilli (singular coccobacillus): Short rods.

4.Spiral: Spiral shaped bacteria with regular or irregular distance between twisting.

Eg. Spirilla and spirochaetes

Fig. 1.5 Morphology of Bacteria

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2. Staining of bacteria

Bacterial staining is the process of coloring of colorless bacterial structural components using stains (dyes).The principle of staining is to identify microorganisms selectively by using dyes, fluorescence and radioisotope emission.

Staining reactions are made possible because of the physical phenomena of capillary osmosis, solubility, adsorption, and absorption of stains or dyes by cells of microorganisms.

Individual variation in the cell wall constituents among different groups of bacteria will consequently produce variations in colors during microscopic examination.

Nucleus is acidic in character and hence, it has greater affinity for basic dyes. Whereas, cytoplasm is basic in character and has greater affinity for acidic dyes.

There are many types of affinity explaining this attraction force:

1.hydrophobic bonding

2.reagent-cell interaction

3.reagent-reagent interaction

4.ionic bonding

5.hydrogen bonding

6.covalent bonding

Why are stains not taken up by every microorganism? Factors controlling selectivity of microbial cells are:

1.number and affinity of binding sites

2.rate of reagent uptake

3.rate of reaction

4.rate of reagent loss (differentiation or regressive staining)

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Properties of dyes

Why dyes color microbial cells?

Because dyes absorb radiation energy in visible region of electromagnetic spectrum i.e., “light”(wave length 400-650). And absorption is anything outside this range it is colorless. E.g., acid fuschin absorbs blue green and transmit red.

General methods of staining 1. Direct staining

Is the process by which microorganisms are stained with simple dyes. E.g., methylene blue

2. Indirect staining – is the process which needs mordants.

A mordant is the substance which, when taken up by the microbial cells helps make dye in return, serving as a link or bridge to make the staining recline possible.

It combines with a dye to form a colored “lake”, which in turn combines with the microbial cell to form a “ cell-mordant-dye- complex”.

It is an integral part of the staining reaction itself, without which no staining could possibly occur. E.g., iodine.

A mordant may be applied before the stain or it may be included as part of the staining technique, or it may be added to the dye solution itself.

An accentuator, on the other hand is not essential to the chemical union of the microbial cells and the dye. It does not participate in the staining reaction, but merely accelerate or hasten the speed of the

26

staining reaction by increasing the staining power and selectivity of the dye.

Progressive staining

-is the process whereby microbial cells are stained in a definite sequence, in order that a satisfactory differential coloration of the cell may be achieved at the end of the

correct time with the staining solution. Regressive staining

-with this technique, the microbial cell is first over stained to obliteratethe cellulare desires, and the excess stain is

removed or decolorized from unwanted part. Differentiation (decolorization)

-is the selective removal of excess stain from the tissue from microbial cells during regressive staining in order that a specific substance may be stained differentiallyh from the surrounding cell.

Differentiation is usually controlled visually by examination under the microscope

Uses

1.To observe the morphology, size, and arrangement of bacteria.

2.To differentiate one group of bacteria from the other group. Biological stains are dyes used to stain micro-organisms.

Types of microbiological stains

. Basic stains

. Acidic stains

. Neutral stains

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NB: This classification is not based on PH of stains.

Basic stains are stains in which the coloring substance is contained in the base part of the stain. The acidic part is colorless. Eg.

Acidic stains are stains in which the coloring substance is contained in the acidic part of the stain. The base part is colorless. It is not commonly used in microbiology laboratory.

Eg. Eosin stain

Neutral stains are stains in which the acidic and basic components of stain are colored.

Neutral dyes stain both nucleic acid and cytoplasm. Eg. Giemsa stain

Types of staining methods

1.Simple staining method

2.Differential staining method

3.Special staining method

1. Simple staining method

It is type of staining method in which only a single dye is used. Usually used to demonstrate bacterial morphology and arrengement Two kinds of simple stains

1. Positive staining: The bacteria or its parts are stained by the dye.

Eg. Carbol fuchsin stain Methylene blue stain Crystal violet stain

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Procedure:

. Make a smear and label it.

. Allow the smear to dry in air.

. Fix the smear over a flame.

.Apply a few drops of positive simple stain like 1% methylene blue, 1% carbolfuchsin or

1% gentian violet for 1 minute.

. Wash off the stain with water.

. Air-dry and examine under the oil immersion objective.

2.Negative staining: The dye stains the background and the bacteria remain unstained. Eg. Indian ink stain Negrosin stain

2.Differential staining method

Multiple stains are used in differential staining method to distinguish different cell structures and/or cell types. Eg. Gram stain and Ziehl- Neelson stain

A.Gram staining method Developed by Christian Gram.

Most bacteria are differentiated by their gram reaction due to differences in their cell wall structure.

Gram-positive bacteria are bacteria that stain purple with crystal violet after decolorizing with acetone-alcohol.

Gram-negative bacteria are bacteria that stain pink with the counter stain (safranin) after losing the primary stain (crystal violet) when treated with acetone-alcohol.

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Required reagents:

. Gram’s Iodine

. Acetone-Alcohol

. Safranin

Procedure:

1.Prepare the smear from the culture or from the specimen.

2.Allow the smear to air-dry completely.

3.Rapidly pass the slide (smear upper most) three times through the flame.

4.Cover the fixed smear with crystal violet for 1 minute and wash with distilled water.

5.Tip off the water and cover the smear with gram’s iodine for 1 minute.

6.Wash off the iodine with clean water.

7.Decolorize rapidly with acetone-alcohol for 30 seconds.

8.Wash off the acetone-alcohol with clean water.

9.Cover the smear with safranin for 1 minute.

10.Wash off the stain wipe the back of the slide. Let the smear to air-dry.

11.Examine the smear with oil immersion objective to look for bacteria.

Interpretation:

. Gram-positive bacterium ……………Purple

. Gram-negative bacterium …………..Pink

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B. Ziehl-Neelson staining method

Developed by Paul Ehrlichin1882, and modified by Ziehl and Neelson

Ziehl-Neelson stain (Acid-fast stain) is used for staining Mycobacteria which are hardly stained by gram staining method. Once the Mycobacteria is stained with primary stain it can not be decolorized with acid, so named as acid-fast bacteria.

Reagents required:

. Carbol-fuchsin

. Acid-Alcohol

. Methylene blue/Malachite green

Procedure for Ziehl-Neelson staining method

1.Prepare the smear from the primary specimen and fix it by passing through the flame and label clearly

2.Place fixed slide on a staining rack and cover each slide with concentrated carbol fuchsin solution.

3.Heat the slide from underneath with sprit lamp until vapor rises (do not boil it) and wait for 3-5 minutes.

4.Wash off the stain with clean water.

5.Cover the smear with 3% acid-alcohol solution until all color is removed (two minutes).

6.Wash off the stain and cover the slide with 1% methylene blue.for one minute.

7.Wash off the stain with clean water and let it air-dry.

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8.Examine the smear under the oil immersion objective to look for acid fast bailli.

Interpretation:

Acid fast bacilli…………..Red

Back ground………………Blue

Reporting system

0 AFB/100 field …………………No AFB seen

1-2 AFB/ 300 field……………….. Scanty

1-10 AFB/100 field………………1+

11-100AFB/100 field.……………2+

1-10 AFB/field…… ……………3+

>10 AFB/field……………… ….4+

NB: AFB means number of acid fast bacilli seen.

3.Special stains

a.Spore staining method

b.Capsule staining method

a. Spore staining method

Procedure:

1.Prepare smear of the spore-forming bacteria and fix in flame.

2.Cover the smear with 5% malachite green solution and heat over steaming water bath for 2-3 minutes.

3.Wash with clean water.

4.Apply 1% safranin for 30 seconds.

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5.Wash with clean water.

6.Dry and examine under the oil immersion objective. b. Capsule staining method: Welch method

Procedure:

1.Prepare smear of capsulated bacteria.

2.Allow smear to air-dry; do not fix the smear.

3.Cover the smear with 1% aqueous crystal violet for 1 minute over steaming water bath.

4.Wash with 20% copper sulfate solution. Do not use water.

5.Dry and examine under the oil immersion objective.

1.5.CULTIVATION OF BACTERIA IN CULTURE MEDIA

Culture media

It is the media containing the required nutrients for bacterial growth. Uses: . Isolation and identification of micro-organisms

. Performing anti-microbial sensitivity tests

Common ingredients of culture media

. Peptone

. Meat extract

. Yeast extract

. Mineral salts

. Carbohydrates

. Agar

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. Water

Peptone: Hydrolyzed product of animal and plant proteins: Free amino acids, peptides and proteoses(large sized peptides).

It provides nitrogen; as well carbohydrates, nucleic acid fractions, minerals and vitamins.

Meat extract: supply amino acids, vitamins and mineral salts.

Yeast extract: It is bacterial growth stimulants.

Mineral salts: these are: Sulfates as a source of sulfur.

. Phosphates as a source of phosphorus.

. Sodium chloride

. Other elements

Carbohydrates: Simple and complex sugars are a source of carbon and energy.

.Assist in the differentiation of bacteria.

Eg. Sucrose in TCBS agar differentiates vibro species. Lactose in MacConkey agar differentiates enterobacteria.

Agar: It is an inert polysaccharide of seaweed. It is not metabolized by micro-organism.

Property

. It has . high gelling strength

. high melting temperature(90-95 oc)

. low gelling temperature

. It forms firm gel at 1.5% W/V concentration.

. It forms semisolid gel at 0.4-0.5% W/V concentration. Uses:

. Solidify culture media

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. May provide calcium and organic ions to inoculated bacteria.

Water

Deionized or distilled water must be used in the preparation of culture media.

Types of culture media

1. Basic /Simple / All purpose media

It is a media that supports the growth of micro-organisms that do not require special nutrients.

Uses :

. To prepare enriched media

. To maintain stock cultures of control bacterial strains

. To subcuture pathogenic bacteria from selective/differential medium prior to performing biochemical or serological tests.

Eg. Nutrient Broth Nutrient Agar

2. Enriched media

Media that are enriched with whole blood, lyzed blood, serum, special extracts or vitamins to support the growth of pathogenic bacteria.

Eg. Blood Agar Chocolate Agar

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3. Enrichment media

Fluid media that increases the numbers of a pathogen by containing enrichments and/or substances that discourage the multiplication of unwanted bacteria.

Eg. Selenite F broth media Alkaline peptone water

4. Selective media

Media which contain substances ( Eg. Antibiotics) that prevent or slow down the growth of bacteria other than pathogens for which the media are intended.

Eg. Modified Thayer –Martin Agar Salmonella-Shigella( SS) agar

1. Differential media

Media to which indicator substances are added to differentiate bacteria.

Eg. TCBS Agar differentiates sucrose fermenting yellow colonies of Vibrio cholerae to non-sucrose fermenting blue colonies other Vibrio species.

NB: Most differential media distinguish between bacteria by an indicator which changes color when acid is produced following carbohydrate fermentation.

2. Transport media

Media containing ingredients to prevent the overgrowth of commensals and ensure the survival of pathogenic bacteria when specimens can not be cultured soon after collection.

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EG. Amies transport media

Stuart media

Kelly-Blair media

Choice of culture media

The selection culture media will depend on:

1.The major pathogens to be isolated, their growth requirements and the features by which they are recognized.

2.Whether the specimens being cultured are from sterile sites or from sites having normal microbial flora.

3.The cost, availability and stability of media.

4.The training and experience of laboratory staff in preparing, using and controlling culture media.

Forms of culture media

1.solid culture media

2.semisolid culture media

3.Fluid culture media

1.solid culture media

. Plate cultures in petri dishes

. stab/slope cultures in tubes and bottles

Uses: Description of bacterial colonies

•size : diameter in mm

•Out line : circular, entire, wavy, indented

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•Elevation: flat, raised, low convex and dome shaped.

•Transparency: transparent, opaque, and translucent.

•Surface: smooth (mucoid) and shiny, rough and dull.

•Color: colorless, white, pink, and pigmented

•changes in medium

Eg. Hemolysis in Blood Agar

Blackening of medium due to hydrogen sulfide production.

2.Semisolid culture media Uses:

. as an enrichment media

. as motility media

3. Fluid culture media

Bacterial growth in fluid media is shown by a turbidity in the medium.

Uses :

. as an enrichment media

. as biochemical testing media

. as blood culture media

Preparation of culture media

Culture media contains essential ingredients for microbial growth requirements.

For successful isolation of pathogens, culture media must be prepared carefully.

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Most culture media are available commercially in ready –made dehydrated form.

The major processes during preparation of culture media

•Weighing and dissolving of culture media ingredients

•Sterilization and sterility testing

•Addition of heat-sensitive ingredients

•Dispensing of culture media

•pH testing of culture media

•Quality assurance of culture media

•Storage of culture media

1.Weighing and dissolving of culture media ingredients

Apply the following while weighing and dissolving of culture media ingredients

•Use ingredients suitable for microbiological use.

•Use clean glass ware, plastic or stainless steel equipment.

•Use distilled water from a glass still.

•Do not open new containers of media before finishing previous ones.

•Weigh in a cool, clean, dry and draught-free atmosphere.

•Weigh accurately using a balance.

•Wear a facemask and glove while weighing and dissolving toxic chemicals.

•Do not delay in making up the medium after weighing.

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•Add powdered ingredients to distilled water and mix by rotating or stirring the flask.

•Stir while heating if heating is required to dissolve the medium.

•Autoclave the medium when the ingredients are dissolved.

2.Sterilization and sterility testing

Always sterilize a medium at the correct temperature and for the correct length of time as instructed in the method of preparation. Methods used to sterilize culture media

A). Autoclaving

B). Steaming to 100 OC

C). Filtration

A)Autoclaving

Autoclaving is used to sterilize most agar and fluid culture media.

B)Steaming at 100 OC

It is used to sterilize media containing ingredients that would be inactivated at temperature over 100 OC and re-melt previously bottled sterile agar media.

C)Filtration

It is used to sterilize additives that are heat-sensitive and can not be autoclaved.

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Sterility testing

The simplest way to test for contamination is to incubate the prepared sample media

At 35-37 OC for 24 hours. Turbidity in fluid media and microbial growth in solid media confirm contamination.

3. Addition of heat-sensitive ingredients

Refrigerated-heat sensitive ingredients should be warmed at room temperature before added to a molten agar medium.

Using an aseptic technique, the ingredients should be added when the medium has cooled to 50 OC, and should be distributed immediately unless further heating is required.

4. pH testing

The pH of most culture media is near neutral, and can be tested using pH papers or pH meter.

5. Dispensing of culture media

Media should be dispensed in a clean draught-free room using aseptic technique and sterile container.

Dispensing agar media in petridish

•Lay out the sterile petridishes on a level surface.

•Mix the medium gently by rotating the flask or bottle.

•Flame sterilize the neck of flask or bottle.

•Pour 15 ml of medium in each petridish.

•Stack the plates after the medium has gelled or cooled.

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•Store the plates in a refrigerator.

NB: Agar plates should be of an even depth and of a firm gel.

The surface of the medium should be smooth and free from bubbles.

6.Quality control

•Inoculate quarter plates of the medium with a five hours broth culture for each control organism.

•Use a straight wire to inoculate and wire loop to spread the inoculum.

•Depending on the species, incubate aerobically, CO2- enriched atmosphere and anaerobically at 35-37 OC for 24

hours.

•Examine for the degree of growth, morphology and other characteristics of microbial colonies.

•Record the result of each control species and compare to your standard reading.

Storage of culture media

•Dehydrated culture media and dry ingredients should be stored at an even temperature in a cool dry place away from direct light.

•Plates of culture media, and additives like serum, blood and antimicrobials in solid form require storage at 2-8 OC.

•Antimicrobials in solution form should be stored at –20 OC.

•All culture media and additives should be labeled with the name and date of preparation.

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Inoculation of culture media

When inoculating culture media, an aseptic technique must be used to prevent contamination of specimens and culture media, and laboratory worker and the environment.

Aseptic technique during inoculation of culture media

•Decontaminate the workbench before and after the work of the day.

•Use facemask and gloves during handling highly infectious specimens.

•Flame sterilize wire loops, straight wires, and metal forceps before and after use.

•Flame the neck of specimen and culture bottles, and tubes after removing and before replacing caps and plugs.

Sterlizing the		Inoculating the fluid
inoculating loop		media with sterilized
with flame		loop

Fig. 1.6 Aseptic inoculation technique

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Inoculation of media in petridishes

The inoculation of media in petridishes is named as ‘plating out’ or ‘looping out’.

Before inoculating a plate of culture media, dry the surface of the media by incubating at 37 OC for 30 minutes.

To inoculate a plate, apply the inoculum to a small area of the plate (‘the well’) using sterile wire loop and then spread and thin out the inoculum to ensure single colony growth.

Fig. 1.7 Methods of ioculating solid culture media in petridishes

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Inoculation of butt and slant media

To inoculate butt and slant media, use a sterile straight wire to stab into the butt and then streak the slant in a zigzag pattern.

Inoculation of slant media

To inoculate slant media, use a straight wire to streak the inoculum down the center of the slant and then spread the inoculum in a zigzag pattern.

Fig. 1.8 Inoculation of slant and bath media

Inoculation of stab media

To inoculate stab media, use a straight wire to stab through the center of the medium and withdraw the wire along the line of inoculum.

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Fig. 1.9 Inoculation of slant media

Inoculation of fluid media

To inoculate fluid media, use straight wire or wire loops.

Incubation of cultures

Inoculated media should be incubated as soon as possible.

Optimal temperature, humidity and gaseous atmosphere should be provided for microorganisms to grow best.

The temperature selected for routine culturing is 35-37 OC.

Some pathogens require CO2-enriched atmosphere to grow in culture media, and the simplest way to provide CO2-enriched atmosphere is to enclose a lighted candle in an airtight jar which provides 3-5% CO2 by the time the candle is extinguished.

Anaerobic atmosphere is essential for the growth of strict anaerobes, and the techniques for obtaining anaerobic conditions are the following:

. Anaerobic jar with a gas generating kit.

. Reducing agents in culture media.

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Fig. 1.10 Co2-enriched atmosphere

1.6. BACTERIAL NUTRITION

Bacteria, like all cells, require nutrients for the maintenance of their metabolism and for cell division.

Bacterial structural components and the macromolecules for the metabolism are synthesized from the elements. The four most important elements of bacteria are carbon, hydrogen, oxygen and nitrogen.

Carbon

Organisms require a source of carbon for the synthesis of numerous organic compounds that comprise protoplast.

Depending on their requirements, bacteria can be classified as

1.Autotrophs: Free-living, non-parasitic bacteria which use carbondioxide as carbon source.

The energy needed for their metabolism can be obtained from:

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. Sun light-Photoautotrophs

. Inorganic compounds by oxidation-Chemoautotrophs

2.Heterotrophs: Parasitic bacteria require more complex organic compounds as their source of carbon and energy.

Human pathogenic bacteria are heterotrophs.

The principal source of carbon is carbohydrate which are degraded either by oxidation, in the presence of oxygen, or by fermentation, in the absence of oxygen, to provide energy in the form of ATP.

Hydrogen and oxygen

•Obtained from water.

•Essential for the growth and maintenance of cell.

Nitrogen

•Constitutes 10% of dry weight of bacterial cell.

•Obtained from organic molecules like proteins and inorganic

molecules like ammonium salts and nitrates.

NB: Main source of nitrogen is ammonia, in the form of ammonium salt.

Growth factors

Growth factors are organic compounds that are required by micro- organisms in small amounts which the cell can not synthesize from other carbon source.

These are aminoacids, purines and pyrimidines, and vitamins. Prototrophs: Wild-type bacteria with normal growth requirements. Auxotrophs: Mutant bacteria, which require an additional growth factor not needed by the parental or wild type strain.

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1.7. BACTERIAL GROWTH

It is an orderly increase in all the components of an organism. It is an increment in biomass.

It is synchronous with bacterial cell reproduction.

Generation time

It is the time taken for the size of a bacterial population to double. Bacteria grow by taking nutrients and incorporate them into cellular components; then bacteria divide into two equal daughter cells and double the number.

Bacterial growth phases

The pattern in cell numbers exhibited by bacterial population obtained after inoculation

Of a bacterium into a new culture medium.

The normal bacterial growth curve has four phases.

1. Lag phase

The period of adaptation with active macro molecular synthesis like DNA, RNA, various enzymes and other structural components.

It is the preparation time for reproduction; no increase in cell number.

2. Exponential(log) phase

The period of active multiplication of cells.

Cell division precedes at a logarithmic rate, and determined by the medium and condition of the culture.

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3. Maximal stationary phase

The period when the bacteria have achieved their maximal cell density or yield.

There is no further increase in viable bacterial cell number. The growth rate is exactly equal to the death rate.

A bacterial population may reach stationary growth when one of the following conditions occur:

1.The required nutrients are exhausted

2.Inhibitory end products are accumulated

3.Physical conditions do not permit a further increase in population size

4.Decline phase

The period at which the rate of death of bacterial cells exceeds the rate of new cell formation.

There is drastic decline in viable cells.

Few organisms may persist for so long time at this period at the expense of nutrients released from dying micro-organisms.

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Fig. 1.11 Bacterial growth curve

Quantitative measurement of bacterial growth

Bacterial growth is measured by determining number of bacteria. The common measuring methods are

1.Viable plate count

2.Direct count

3.Turbidimetric method

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1. Viable plate count

The most common method of estimating bacterial growth which involves counting the number of bacterial colonies grown on solid media after incubation of the inoculated media for 18-24 hours.

Procedure

•The sample is serially diluted.

•The suspension is inoculated on solid media by surface spread technique i.e. the suspension is spread

•The plate is incubated for 18-24 hrs to allow the bacteria to grow and form colonies.

•The concentration of bacteria in the original sample can be determined by counting the visible colonies multiplied by the dilution factor.

Number of colonies =Number of colonies Χ dilution factor Volume of sample

NB: The statistically significant plate count is between 30 and 300 colonies.

Less than 30 colonies on a plate are not accepted for statistical reasons.

Greater than 300 colonies on a plate are too close to distinguish as an individual colony forming unit (too numerous to count).

Limitation of viable plate count: It selectively in favor of a certain group of bacterial population.

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2. Direct count

It involves direct microscopic counting of bacteria in the sample using counting chamber.

It is relatively quick and does not need the sample to be incubated.

Procedure

. Serial dilution of the sample

. Fill known area and volume of the counting chamber with the sample

. Total number of bacteria in the sample per unit volume is equal to No. of bacteria in the sample Χ the No. of squares Χ dilution factor.

3. Turbidimetric method

It is the method of determination of bacterial growth in liquid media. Bacterial growth increases the turbidity of liquid to absorb light.

The turbidity of the suspension is determined by spectrophotometer.

Factors influencing bacterial growth in vitro

Not all bacterial species grow under identical environmental conditions. Each bacterial species has a specific tolerance range for specific environmental parameters.

Out side the tolerance range environmental conditions for a bacteria to reproduce, it may survive in dormant state or may lose viability. Rates of bacterial growth are greatly influenced by the following environmental parameters.

. Nutrition

. Temperature

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. Oxygen

. PH

. Salinity

. Pressure

. Light radiation

1. Nutrition

The following nutrients must be provided for optimal bacterial growth.

•Hydrogen donors and acceptors

•Carbon source

•Nitrogen source

•Minerals: sulfur and phosphorus, trace elements

•Growth factors: amino acids, purines, pyrimidines and vitamins.

2.Temperature

Temperature tolerance range: The minimum and maximum temperature at which a micro-organism can grow; which is different in different species of bacteria.

Optimal growth range of temperature: The temperature at which the maximum growth rate occurs; and results in the shortest generation time of bacteria.

Based on different optimal growth temperature requirement, bacteria are divided into:

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Optimal growth temperature

. Psychrophilic bacteria	.15-20 o c; grow best at low T0 range
. Mesophilic bacteria	.30-370c; grow best at middle T0 range
. Thermophilic bacteria	.50-60oc; grow best at high T0 range

NB: Most human pathogens and many of the normal flora of human bodies have an optimal temperature of 370c; There fore they are mesophilic bacteria.

3. Oxygen

Base on oxygen requirements and tolerance, bacteria are divided classified as:

. Obligate aerobes

. Obligate anaerobes

. Facultative anaerobes

. Microaerophiles

•Obligate aerobic bacteria grow only when free oxygen is available to support their respiratory metabolism.

They obtain ATP by using oxygen as a final electron acceptor in respiration.

•Obligate anaerobic bacteria grow in the absence of oxygen; exposure to oxygen kills anaerobes.

•Facultative anaerobic bacteria grow in the presence or

absence of oxygen.

They obtain ATP by fermentation or anaerobic respiration

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•Microaerophilic bacteria grow best at reduced oxygen tension; high oxygen tension is toxic to them.

4.Hydrogen ion concentration

It is a measure of acidity and alkalinity.

PH<7 is acidic

PH =7 is neutral

PH>7 ia alkaline

•Neutrophilic bacteria grow best at near neutral PH value.

•Acidicophilic bacteria prefer to grow at low PH value (acidic medium).

•Alkalinophilic bacteria prefer to grow at high PH value (alkaline medium).

•Most pathogenic bacteria grow best at PH of 6-8.

5.Salinity

Salt content of the medium affects bacterial growth. Halophilic bacteria grow best at high salt concentration.

. Moderate halophiles require 3% salt concentration.

. Extreme halophiles require 15% salt concentration.

Most bacteia can not tolerate high salt concentration. High salt concentration disrupts membrane transport systems and denatures proteins of bacteria but halophiles have adaptive mechanisms to tolerate high salt concentration.

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6. Pressure

Osmotic pressure: The pressure exerted on bacterial cell surface as a result of difference in solute concentration between the inside and out side of a cell.

Osmotolerant bacteria can grow in solutions with high solute concentration.

Osmophilic bacteria grow best at high hydrostatic pressure. Hydrostatic pressure: The pressure exerted by the weight of a water column.

High hydrostatic pressures more than 200 atmosphere generally inactivates enzymes and disrupts membrane transport process. Barotolerant bacteriacan grow at high hydrostatic pressure. Barophilic bacteria grow best at high hydrostatic pressure.

7. Light radiation

Photosynthetic bacteria require light in the visible spectrum to carry out photosynthesis.

COMMON BIOCHEMICAL TESTS

Litmus milk reduction test:

Required: Litmus milk medium Wire loop

Bunsen burner Test bacteria

Method:

. Inoculate 0.5 ml of sterile litmus milk medium with the test bacteria.

. Incubate at 35-37 Oc for up to 4 hrs.

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. Observe for changes in color every 30 min.

Results:

. Change in color of medium from pink to white or pale is suggestive of enterococci

CAMP test (Christie, Atkins , Munich Paterson )

Principle: S.agalaciae produce protein named as camp factor, which interacts with staphylococci β- hemolysin on sheep red blood cell.

Method:

. Streak S.aureus isolate across sheep blood agar plate.

. Inoculate the test bacteria at right angle to staphylococci with out touching it.

. Incubate over night at 35-37 Oc.

. Formation of an arrow-head shaped area of hemolysis indicates interaction of camp factor with staphylococci hemolysin.

Bacitracin test

Principle: Streptococcus pyogenes is sensitive to bacitracin but other kinds of streptocci are resistant to bacitracin.

Method:

. Streak a blood agar plate with the isolated organism.

. Place bacitracin disc in the streaked area.

. Incubate the plate for 24 hours at 37 0c.

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. Examine the plate for a zone of no-growth around the disc.

No-growth around the disc...…………… S.pyogenes

Growth around the disc ………………… Other streptococci

Optochin test

Principle: S. pneumoniae is sensitive to optochin disc unlike other alpha-hemolytic streptococci.

Method:

. Streak a blood agar plate with the isolated organism.

. Place optochin disc in the streaked area.

. Incubate the plate for 24 hours at 37 0c.

. Examine the plate for a zone of no-growth around the disc.

No-growth around the disc ..…S.pneumoniae

Growth around the disc …otherAlpha-

hemolytic streptococci

Carbohydrate utilization test

Method:

. Prepare saline suspension of test bacteria

. Add 0.1ml of bacterial suspension into each of four test tubes containing glucose, lactose, maltose and sucrose carbohydrate discs.

. Incubate in a water bath at 37Oc and examine at 30 min intervals for 5 hrs for change in color.

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Result: Change in color from red to yellow-orange indicates carbohydrate utilization.

BILE SOLUBILITY TEST

This helps to differentate S.pneumoniae, which is soluble in bile and bile salts, from viridans streptococci which are insoluble.

Principle

A heavy inoculum of the test organism is emulsified in physiological saline to give a turbid suspension. The bile salt sodium deoxycholate is then added. The test can also be performed by adding the bile salt to a broth culture of the organism. The bile salt dissoles S.pneumoniae as shown by a clearing of the turbidity within 10-15 minutes. Viridans streptococci are not dissolved and therefore there is no clearing of the turbidity.

Required

Sodium deochollate100g/l

Physiological saline (sodium chloride, 8.5g/l)

Method

•Emulsify several colonies of the test organism in a tube containing 2ml of sterile physiological saline, to give a turbid suspension.

•Divide the organism suspension between two tubes.

•To one tube, add 2 drops of the sodium deoxycholate reagent and mix.

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•To the other tube, add 2 drops of sterile distilled water and mix.

•Leave both tubes for 10-15 minutes.

•Look for a clearing of turbidity in the tube containing the sodium deoxycholate.

Results
Clearing of turbidity --------------	Probably
	S.pneumoniae
No clearing of turbidity -----------	organism is probably
	Not S.pneumoniae

There should be no clearing of turbidity in the tube to which distilled water was added. If there is, repeat the test.

Note: Some strains of S.pneumoniae are not dissolved by bile salts, and very occasionally some strains of viridans streptococci give a positive tests.

Controls

Bile solubility positive control: Streptococcus pneumoniae.

Bile solubility negative control: Streptococcus faecalis.

CATALASE TEST

This test is used to differentiate those bacteria that produce the enzyme catalase, such as staphylococci, from non-catalase producing bacteria such as streptococci.

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Principle

Catalase acts as a catalyst in the breakdown of hydrogen peroxide to oxygen and water.

An organism is tested for catalase production by bringing it into contact with hydrogen peroxide. Bubbles of oxygen are released if the organism is a catalase producer. The culture should not be more than 24 hours old.

Care must be taken if testing an organism cultured on a medium containing blood because catalase is present in red cells. If any of the blood agar is removed with the colony, a false positive reaction will occur. It is usually recommended, therefore, that catalase testing be performed from a blood free culture medium such as nutrient agar.

Required

a.Hydrogen peroxide, 3% H2O2

Note: Shaking the reagent before use will help to expel any dissolved oxygen. False positive reactions may occur if the hydrogen peroxide contains dissolved oxygen.

Method

•Pour 2-3ml of the hydrogen peroxide solution into a test tube.

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•Using a sterile wooden stick or a glass rod, remove a

good growth of the test organism and immerse it in the hydrogen peroxide solution.

Note: A nichrome wire loop must not be used because this may give a false positive reaction.

•Look for immediate bubbling.

Results
Active bubbling -----------------	Positive test
	Catalase produced
No release of bubbles ----------	Negative test
	No catalase produced

Note: if the organism has been cultured on an agar slope, pour about 1ml of the hydrogen peroxide solution over a good growth of the organism, and look for the release of bubbles.

Caution: performing the test on a slide is not recommended because of the risk of contamination from active bubbling.

If the rapid slide technique is used, the hydrogen peroxide solution should be added to the organism suspension after placing the slide in a petridish. The dish should then be covered immediately, and the preparation observed for bubbling through the lid.

Controls

Positive catalase control: Staphylococcus species.

Negative catalase control: Streptococcus species.

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CITRATE UTILIZATION TES

This test is one of several techniques used to assist in the identification of enterobacteria. The test is based on the ability of an organism to use citrate as its only source of carbon and ammonia as its only source of nitrogen.

Principle

The test organism is cultured in a medium which contains sodium citrate, an ammonium salt, and the indicator bromo – thymol blue. Growth in the medium is shown by turbidity and a change in colour of the indicator from light green to blue, due to the alkaline reaction, following citrate utilization.

Required

Koser’s citrate medium or Simmon’s citrate agar.

Method

Using a sterile straight wire, inoculate 3-4ml of sterile Koser’s citrate medium with a broth culture of the test organism.

Note: Care must be taken not to contaminate the medium with carbon particles, such as from a frequently flamed wire.

Incubate the inoculated broth at 35 – 37OC for up to 4 days, checking daily for growth.

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Results
Turbidity and blue colour	------------------------------------ Positive test
	Citrate
utilized
No growth ----------------------------------------------	Negative test
	Citrate not utilized

Controls

Positive citrate control: Klebslella pneumonlae

Negative citrate control: Escherichia coli.

COAGULASE TEST

This test is used to differentiate Staphylococcus aureus which produces the enzyme coagulase, from S.epidermidis and S.saprophyticus which do not produce coagulase.

Principle

Coagulase causes plasma to clot by converting fibrinogen to fibrin. Two types of coagulase are produced by most strains of S.aureus:

Free coagulase which converts fibrinogen to fibrin by activating a coagulase – reacting factor present in plasma. Free coagulase is detected by the appearance of a fibrin clot in the tube test.

Bound coagulase (clumping factor) which converts fibrinogen directlyto fibrin without requiring a coagulase – reacting factor. It can be detected by the clumping of bacterial cells in the rapid slide test. It is usually recommended that a tube test should be performed on all negative slide tests. A tube test must always be

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performed if the result of the slide test is not clear, or when the slide test is negative and the Staphylococcus has been isolated from a serious infection.

Required

a.Undiluted human plasma (preferably pooled) or rabbit plasma. The plasma should be allowed to warm to room temperature before being used.

Plasma from EDTA (ethylenediamine – tetra – acetic acid) or citrate anticoagulated blood is usually used. Note: Occasionally citrate-utilizing organisms such as Klebsilla can cause the clotting of citrated plasma in the tube test. This can be prevented by adding heparin to the citrated plasma. It is also possible for human plasma to contain inhibitory substances which can interfere with coagulase testing. Adequate controls must be included for both slide and tube tests.

Method for slide test (to detect bound coagulase)

Place a drop of physiological saline on each end of a slide, or on two separate slides.

Emulsiy a colony of the test organism in each of the drops to make two thick suspensions.

Note: Colonies from a mannitol salt agar culture are not suitable for coagulase testing. The organism must first be cultured on nutrient agar or blood agar.

Add a drop of plasma to one of the suspensions, and mix gently. Look for clumping of the organisms within 10 seconds.

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No plasma is added to the second suspension. This is used to differentiate any granular appearance of the organism form true coagulase clumping.

Results
Clamping within 10 secs ------------------------	S.aureus
No clumping within 10 secs-----------	No bound coagulase produced.

Controls

Positive coagulase control: Staphylococcus aureus.

Negative coagulase control: Escherichia coli or Staphylococcus epldermids

Method for tube test (detect free coagulase)

Dilute the plasma 1 in 10 in physiological saline (mix 0.2 ml of plasma with 1.8ml of saline).

Take three small test tubes and label:

T = Test organism (18-24h broth culture)

Pos = Positive control (18-24h staph. Aureus broth culture)

Neg = Negative control (sterile broth) A suitable broth is brain heart infusion

Pipette 0.5ml of the diluted plasma into each tube.

Add 5 drops (about 0.1ml) of the test organism culture to the tube labeled ‘T’.

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Add 5 drops of the staph. Aureus culture to the tube labeled ‘Pos’.

Add 5 drops of sterile broth to the tube labeled ‘Neg’.

After mixing gently, incubate the three tubes at 35-37OC. Examine for clotting after 1 hour. If no clotting has occurred, examine at 30minute intervals for up to 6 hours.

When looking for clotting, gently tilt each tube.

Most Staph, aureus strains produce a fibrin clot within 1 hour of incubation. There should be no fibrin clot in the negative control tube.

Results
Fibrin clot -----------------------------	S. aureus
No fibrin clot -------------------------	No free coagulase produced

DEOXYRIBONUCLEASE (DNAse) TEST

This test is used to differentiate Staph. Aureus which produces the enzyme DNAse from other staphylococci which do not produce DNAse. It is particularly useful if plasma is not available to peform a coagulase test or when the results of a coagulase test are difficult to interpret.

Principle

Deoxyribonuclease hydrolyzes deoxyribonucleic acid (DNA).

The test organism is cultured on a medium which contains DNA. After overnight incubation, the colonies are tested for DNAse production by flooding the plate with a weak hydrochloric acid solution. The acid precipitates unhydrolyzed DNA. DNAse producing

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colonies are, therefore surrounded by clear areas indicating DNA hydrolysis.

Required

a.DNAse agar plate

Up to six organisms may be tested on the same plate.

b.Hydrochloric acid, 1 mol/l

Method

Divide a DNAse plate into the required number of strips by marking the underside of the plate.

Using a sterile loop or swab, spot – inoculate the test and control organisms. Make sure each test area is clearly labeled. Incubate the plate at 36-37OC overnight.

Cover the surface of the plate with 1mol/l hydrochloric acid solution. Tip off the excess acid.

58.Look for clearing around the colonies within 5minutes of adding the acid.

Results
Clearing around the colonies -----------------	DBAse positive strain.
No clearing around to colonies -------------	DNAse negative strain.

Controls

Positive DNAse control: staphylococcus arureus .

Negative DNAse control: staphylococcus epidermidis.

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HYDROGEN SULPHID (H2S) PRODUCTION

The detection of hydrogen sulphide gas (H2S) is used mainly to assist in the identification of enterobacteria and occasionally to differentiate other bacteria such as Bacteroides and Bruceila species. H2S is produced when sulphur – containing amino acids are decomposed.

Use of Kligler iron agar (KIA) to detect H2S

This medium is suitable for detecting H2S production by enterobacteria. H2S is detected by the ferric citrate contained in the medium.

Inoculate the test organism into KIA and incubate it at appropriate temperature over night.

Observe blacking of the medium

Lead acetate paper test to detect H2S

When a sensitive technique for detecting H2S production is required, the lead acetate paper test is recommended.

Inoculate a tube or bottle of sterile peptone water or nutrient broth with the test organism.

Insert a lead acetate paper strip in the neck of the bottle or tube above the medium, and stopper well.

Incubate the inoculated medium at 35-37OC, and examine daily for a blackening of the lower part of the strip.

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Results
Blackening -----------------------------	Positive test H2S produced
No blackening -------------------------	Negative test No H2S
produced.

Controls

Positive hydrogen sulphide control: proteus vulgaris

Negative hydrogen sulphide control: shigella species

INDOLE TEST

Testing for indole production is important in the identification of enterobacteria. Most sftrains of E.coli, P.vulgaris, P.rettgeri, M.morganll, and providencia species break down the amino acid tryptophan with the release of indole.

Principle

The test organism is cultured in a medium which contains tryptophan. Indole production is detected by Kovac’s or Ehrlich’s reagent which contains 4(P)-dimethylaminobenzaldehyde. This reacts with the indole to produce a red coloured compound. In the following method the use of the combined motility indole urea (MIU) medium is described. A Kovac’s ragent paper strip is inserted in the neck of the tube, and indole production is indicated by a reddening of the strip. Indole is a volatile substance (easily vaporized). The tube must be well stoppered during incubation.

The indole test can also be performed by culturing the organism in tryptone water or peptone water containing tryptophan, and

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detecting indole production by adding Kovac’s or Ehrlich’s reagent to an 18-24h culture.

Required

Motility indole urea (MIU) medium

MIU medium indicates whether an organism is motile or non-motile, indole positive or negative, and urease positive or negative.

Method

Using a sterile straight wire, inoculate 5ml of sterile MIU medium with a smooth colony of the test organism.

Place an indole paper strip in the neck of the MIU tube above the medium, and stopper the tube, incubate at 35-37OC overnight.

Examine for idole production by looking for a reddening of the lower part.

Results
Reddening of strip -----------------------------	Positive test
	Indoloe produced
Noered colour ----------------------------------	Negative test
	No Indoloe produced
Note: If the reaction is weak, confirm the result by adding 1ml of
Kovac’s regent to the culture. Examine for a red colouring of the
surface layer within 10 minutes.
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Controls

Positive indole control: Escherichia coli

Negative indoloe control: Enterobacter aerogenes.

Motility Test

This is shown by a spreading turbidity from the stab line or a turbidity throughout the medium (compare with an uninoculated tube).

Urease production

This is shown by a red-pink colour in the medium.

NITRATE REDUCTION TEST

This test is used to differentiate members of the Enterobacteriaceae that produce the enzyme nitrate reductase, from Gram negative bacteria that do not produce the enzyme.

The test is also helpful in differentiating Mycobacterium species as explained.

Principle

A heavy inoculum of the test organism is incubated in a broth containing nitrate. After 4 hours, the broth is tested fro the reduction of nitrate to nitrite by adding sulphanilic acid reagent. If nitrite is present, the acid reagent is diazotizex and forms a pink-red compound with alpha-naphthylamine. When nitrite is not detected it

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is necessary to test whether the organism has reduced the nitrate beyond nitrite. This is done indirectly by checking whether the broth still contains nitrate. Zinc dust is added which will convert any nitrate to nitrate. If no nitrite is detected when the zinc dust is added, it can be assumed that all the nitrate has been reduced beyond nitrite to nitrogen gas or ammonia by a nitrate reducing organism.

Required

a.Nitrate broth

b.Sulphanilic acid reagent

c.Alphanaphthylamine reagent

d.Zinc dust

Method

Inoculate 0.6 ml of sterile nitrate broth with a heavy growth of the test organism.

Incubate at 35-37OC for 4 hours.

Add 1 drop of sulphanilic acid reagent and 1 drop of alphanaphthylamine reagent.

Shake to mix and look for a red colour.

Results
Red colour	----------------------------- Positive test
	Nitrate reduced
If no red colour is produced, add a very small amount (knife point) of
zink dust powder. Look again for a red colour and intrpret as follows:
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Red colour -----------------------------	Negative test
	No reduction of nitrate
No red colour -------------------------	Positive test
	Nitrate reduced

Controls

Positive nitrate reduction control: Escherichia coli. Negative nitrate

reduction control: Pseudomonas aeruginosa.

OXIDASE TEST (Cytochrome Oxidase)

The oxidase test is used to assist in the identification of pseudomonas, Neisseria, Vibrio, and Pasteurella species, all of which produce oxidase enzymes.

Principle

A piece of filter paper is soaked with a few drops of oxidase reagent. A colony of the test organism is then smeared on the filter paper. If the organism is oxidase - producing, the phenylenediamine in the reagent will be oxidized to a deep purple colour. Occasionally the test is performed by flooding the culture plate with oxidase reagent but this technique is not recommended for routine use because the reagent rapidly kills bacteria. It can be useful, however, when attempting to isolate N.gonorrhoeae colonies from mixed cultures in the absence of a selective medium. The oxidase positive colonies must be removed and subcultured within 30 seconds of flooding the plate.

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Important: Acidity inhibits oxidase enzyme activity. The oxidase test must not be performed, therefore, on colonies that produce fermentation on carbohydrate – containing media, such as sucrose fermenting V.cholerae colonies on TCBS medium, Subinoculation on nutrient agar is required before the oxidase test can be performed reliably. Non – fermenting colonies, however, can be tested. Colonies tested from a medium that contains nitrate may give unreliable oxidase test results.

Required

−Oxidase reagent

Freshly prepared

This is a 10g/l solution of tetramethyl –p-phenylenediamine dihydrochloride.

Note: Oxidase reagent is easily oxidized. When oxidized, it is blue in colour and must not be used.

Method

Place a piece of filter paper in a clean petri dish and add 2 or 3 drops of freshly prepared oxidase reagent.

Using a piece of stick or glass rod (not an oxidized wire loop), remove a colony of the test organism, and smear it on the filter paper.

Look for the development of a blue – purple colour within a few seconds.

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Results
Blue-purple colour -------------------------------------	Positive test
(within 10 seconds)	Oxidase produced
No blue – purple colour -------------------------------	Negative test
(within 10 seconds)	No oxidase produced

Note: ignore any blue – purple colour that develops after 10 seconds.

Controls

Positive oxidase control: Pseudomonas aeruginosa.

Negative oxidase control: Escherichia coli.

OXIDATION – FERMENTATION (O-F) TEST

This test is used to differentiate those organisms that oxidize. Carbohydrates (aerobic utilization) Such as Pseudomonas aeruginosa, from those organisms that ferment carbohydrates (anaerobic utilization) such as members of the Entero- bacteriaaceae.

Principle

The test organism is inoculated into two tubes of a tryptone or peptone agar medium containing glucose (or other carbohydrate) and the indicator bromothymol blue. The inoculated medium in one tube is sealed with alayer of liquid paraffin to exclude oxygen.

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Fermentative organisms utilize the carbohydrate in both the open and sealed tubes and the colour of the medium changes from green to yellow. Oxidative organisms, however, are able to use the carbohydrate only in the open tube. There is no carbohydrate utilization in the sealed tube (medium remains green).

Although most genera of aerobic bacteria are either carbohydrate oxidizers or fermenters, the production of acid may be slow and therefore cultures are usually incubated for 7-14 days.

Required

a.Oxidation fermentation (O-F) medium

Glucose, maltose, and sucrose O-F media are the most commonly used.

b.Sterile paraffin oil (liquid paraffin)

Method

Using a sterile straight wire, inoculate the test organism to the bottom of two bottles (or more if testing several carbohydrates) of sterile O-F medium. Use a heavy inoculum.

Cover the incculated medium in one of the tubes (or one from each carbohydrate pair) with a 10mm deep layer of sterile paraffin oil or molten wax.

Incubate the tubes at 35-37OC for up to 14 days. Examine daily for carbohydrate utilization as shown by acid production.

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Results

Open	Sealed	Interpretation
tube	tube
Yellow	Green	Oxidative organism
Yellow	Yellow	Fermentative organism
Green or blue	Green	No utilization of carbohydrate

Controls

Oxidative control: Pseudomonas aeruginosa.

Fermentative control: Escherichia coli.

PHENYLALANINE DEAMINASE TEST

The test, which is also referred to as the Phenylpyruvic acid (PPA) test, is used mainly to assist in the identification of enterobacteria. It is based on the ability of bacteria such as Proteus specdies and some Providencia strains to break down phenylalanine (by oxidative deamination) with the production of phenylpyruvic acid. Y.enterocolitica (urease – producer is a phenylalanine negative.

Principle

The test organism is cultured on a slope of phenyiaianine medium. After overnight incubation, the deamination of phenylaianjine to phenyl – pyruvic acid is detected by adding iron III chloride (ferric chloride) which produces a green colour on the surface of the culture.

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Required

a.Phanylalanine agar

b.Iron III chloride (ferric chloride),

100g/l (10% w/v). The reagent must be freshly prepared.

Method

. inoculate a slope of phenylalanine agar with the test organism, and incubate at 35-37Oc overnight.

. Add 4 or 5 drops of the freshly prepared iron III chloride reagent to the culture, allowing the reagent to run down the slope.

. Look for a green colour on the slope.

Results
Green colour ------------------------------------	Positive test
(Within 5 minutes)	Phenylanine deaminated
No green colour ---------------------------------	Negative e test
	No deamination of phenylalanine

Controls

Positive PPA control: Proteus species

Negative PPA control: Escherichia coli.

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TWEEN 80 HYDROLYSIS TEST

This test is used mainly to differentiate slow–growing Mycobacterium species as described in 44:1 species that hydrolize the detergent Tween 80 with the production of oleic acid are listed in Chart.

Principle

The test organism is incubated in a Tween 80 buffered substrate that contains the indicator neutral red. Tween hydrolysis is detected by a change in colour of the indicator from amber to pink – red due to the production of oleic acid.

Required

Tween 80 phosphate buffered substrate with neutral red. *The substrate requires storage at 4Oc

Method

. Inoculate 4 ml of sterile Tween 80 phosphate buffered substrate with a loopful of growth of the test organism.

. Incubate at 35-37 Oc for up to 18 days. Examine at 5,10, and 18 days for a change in colour of the substrate from amber to pink-red, as shown in colour.

Results
Pink-red substrate --------------------------------------	Positive test
	Tween 80 hydrolyzed
No change in colour ------------------------------------	Negative test
	No hydrolysis of Tween 80
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Controls

Positive Tween hydrolysis control: Mycobacterium kansasii.

Negative Tween hydrolysis control: Use an unlnoculated tube of substrate.

UREASETEST

Testing for urease enzyme activity is important in differentiating entrobacteria. Proteus strains are strong urease producers. Y.enterocolitica also shows urease activity (Weakly a 35-37 Oc) Salmonellae and shigellae do not produce urease.

Principle

The test organism is cultured in a medium which contains urea and the indicator phenol red. If the strain is urease-producing, the enzyme will beak down the urea (by hydrolysis) to give ammonia and carbon diaoxide. With the release of ammonia, the medium becomes alkaline as shown by a change iin colour of the indicator to red-pink.

The method described is that which uses the combined motility indole urea (MIU) medium, urease production can also be detected by culturing the organisms in Christensen’s urea broth.

Required

a. Motility indole urea (MIU) medium.

Method

. Using a sterile straight wire, inoculate a tube of sterile MIU medium with a smooth colony of the test organism.

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. Place an indole paper strip in the neck of the MIU tube above the medium. Stopper the tube and incubate at 35- 37 Oc overnight.

. Examine for urease production by looking for a red- pink colour in the medium as shown in colour.

Results
Red-pink medium----------------------------------------	Positive test
	Urease produced
No red-pink colour -------------------------------------	Negative test
	No urease produced

Controls

Positive urease control: Proteus vulgaries.

Negative urease control: Eshcherlchle coli.

Note: After overnight incubation, y.enterocolitica gives a weak urease reaction and is non-motile at 35-37 Oc. At room temperature (22-293Oc), the species is motile and shows a stronger urease reaction.

VOGES – PROSKAUER (V-P) TEST

This test is occationally used to assist in the differentiation of enterobacteria. K.pneumoniae, Vibrio cholerae blovar el tor, and some strains of Enterobacter, ferment glucose with the production of acetylmethylcarbinol (acetoin) which can be detected by an oxidation reaction.

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Principle

The test organism is cultured in a glucose phosphate peptone water for 48 hours. Sodium hydroxide and a small amount of creatine are then added. Under alkaline conditions and exposure to the air, the acation produced from the fermentation of the glucose is oxidized to diacetyl which forms a pink compound with the creatine.

Required

a.Glucose phosphate peptone water.

b.Sodium hydroxide, 400g/l.

c.Creatine poweder.

Method

. Inoculate 2ml of sterile glucose phosphate peptone water with the test organism. Incubate at 35-37 Oc for 48hours.

. Add a very small amount (knife point)of creatine and mix.

. Add about 3ml of the sodium hydroxide reagent and shake well,

Caution: The sodium hydroxide reagent is corrosive, therefore handle with care and do not mouth – pipette.

. Remove the bottle cap, and leave for 1 hour at room temperature. Look for the slow development of a pink – red.

Results
Pink – red colour --------------------------------------	Positive test
	Acetoine produced
No pink – red colour ----------------------------------	Negative test
	No acetoin produced
	84

Controls

V-P Positive control: Enterobacter aerogenes or

Klebsiella pneumoniae

V-P Negative control: Escherichia coli.

1.8. BACTERIAL GENETICS

Genetics is the study of inheritance. Bacterial inherited characteristics are encoded in DNA.

Bacteria have two types of DNA that contain their genes. These are :

. Chromosome

. Extra chromosome: Plasmid

The bacterial chromosome is circular, double stranded DNA attached to bacterial cell membrane.

DNA replication in bacteria is semi-conservative i.e. each strand of DNA is conserved intact during replication and becomes one of the two strands of the new daughter molecules.

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Fig. 1.12 Bacterial chromosome

Plasmids are self-replicating extra chromosomal DNA molecules. It multiplies independent of the host cell.

Multiple copies of the same plasmid may be present in each bacterial cell.

Different plasmids are also often present in the same bacterial cell.

Plasmid types

There are many types of plasmid types. The following are examples.

a.R factors: Plasmids which contain genes that code for antibiotic resistance.

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b. Col factors: Plasmids which contain genes that code for extracellular toxin (colicines) production that inhibit strains of the same and different species of bacteria.

c.F(fertility) factors: Plasmids that can recombine itself with the bacterial chromosome.

It promotes transfer of the chromosome at a high frequency of recombination into the chromosome of a second (recipient) bacterial cell during mating.

Genetic variation in Bacteria

Mechanisms: Mutation and Gene transfer

1.Mutation: It is due to a chemical alteration in DNA.

It could be spontaneous or induced by chemical and physical meanses

Mutants are variants in which one or more bases in their DNA are altered; which are heritable and irreversible

Types of mutation

1.Substitution: Change of a single base.

2.Deletion: Los of a base.

3.Insertion: Addition of a base.

2. Gene transfer

There are three types of gene transfer that alter the DNA gene content of bacteria.

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These are:

. Transformation

. Transduction

. Conjugation

1.Transformation occurs when fragments of exogenous bacterial DNA are taken up and absorbed into recipient bacterial cells. Transformation of genes from one bacterium to another results in

. Change in pathogenicity of the bacterium.

. Change in antibiotic sensitivity pattern of bacterium.

Competence: The recipient bacterium must be competent to absorb the exogenous fragments of bacterial DNA.

Frequency: The frequency of transformation is low.

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Fig. 1.13 Transformation; gene transfer by the uptake & subsequent

recombination of a fragment of exogenous bacterial DNA

2.Transduction occurs when fragments of chromosomal DNA is transferred or transduced into a second bacterium by phage.

During phage replication, the bacterial DNA may be accidentally enclosed instead of the normal phage DNA, and when this particle which enclosed the bacterial DNA infects a second bacterial cell, the DNA from the first bacterium is released and incorporated into The chromosome of the second bacterium.

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3.Conjugation occurs when plasmid DNA is transferred from donor to recipient bacterium by direct contact via a sex pilus.

Fig. 1.15 Conjugation; plasmid gene transfer by conjugation

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4. Transposition

Mechanism which enhances genetic flexibility among plasmids and bacterial chromosomes. Transposons(Jumping genes) are segments of DNA that can transpose or move extremely readily, from plasmid to plasmid or from plasmid to chromosome(and viceversa).In this way, plasmid genes become part of the chromosomal component of genes.

When transposons transfer to a new site, it is usually a copy of the transposon that moves, the original transposon remaining in situ.

Transposons code for toxin production, resistance to antibiotics as wellas other fuctions.

1.9. STERILIZATION AND DISINFECTION

Sterilization: Destruction of all forms of microbial life including spores.

Disinfection: Destruction of microbes that cause disease; may not be effective in killing spores.

Antisepsis: destruction or inhibition of microorganisms in living tissue there by limiting or preventing the harmful effect of infection. Sterilizing and disinfecting agents are divided into two groups.

These are:

1.Chemical methods of sterilization and disinfection

2.physical methods of sterilization and disinfection

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1.9.1. Chemical methods of sterilization and disinfection

These chemical agents destroy any type of microbes with out showing any form of selectivity unlike antibiotics.

The efficacy of these agents depends on the following factors.

1.Concentration of the agent

There is a relationship between the concentration of the agent and the time required to kill a given fraction of the microbial population.

2.Time of exposure

Microbes are killed with a reasonable length of time with chemical agents.

3.pH of the medium where action is to take place

Hydrogen ion concentration determines degree of ionization of the chemical and bacterial surface charge.

The non-ionized form passes through the bacterial cell membrane more readily than the ionized form.

4.Temperature

Bactericidal potency of the chemical agent increases with an increase in temperature.

An increase in 100c doubles the bacterial death rate.

5.Nature of the organism

. Species of the bacteria

. Growth phase of bacteria in culture

. Presence of capsule, spore and other special structures

. Number of bacteria in test system

6.Presence of extraneous materials

Organic materials like serum, blood or pus makes chemicals inert that are highly active in their absence.

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Classification of chemical methods of sterilization and disinfection

1.Chemical agents that damage the cell membrane

. Surface active agents

. Phenols

. Organic solvents

2.Chemical agents that denature proteins

. Acids and alkalies

3.Chemical agents that modify functional groups of proteins and nucleic acids

. Heavy metals

. Oxidizing agents

. Dyes

. Alkylating agents

1. Chemical agents that damage the cell membrane

Surface active agents

a.Cationic agents

.Quaternary ammonium compounds (Quates)

It causes loss of cell membrane semi permeability leading to loss of nutrients and essential metabolites. It as well denatures protein.

•More active in Gram-positive bacteria than in Gram- negative bacteria.

•More active at alkaline PH

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•Inactivated by organic materials.

b.Anionic agents

.Soaps and fatty acids

It causes gross disruption of cell membrane lipoprotein frame work.

. More active in Gram-positive bacteria than in Gram-negative bacteria.

. Active at acidic PH

Phenolic compounds

Phenol is highly effective in Gram positive bacteria. Clinically no more used because of its neurotoxic effect.

Currently used as a standard for measuring bactericidal potency of new chemicals i.e. phenol coefficient.

Phenol coefficient is the ratio of the concentration of the new chemical agent being tested to the concentration of the reference standard (phenol) required to kill in a specific time.

If phenol coefficient is less than one, the new chemical agent is less effective than phenol.

If phenol coefficient is equal to one, the new chemical agent is equal to phenol in efficacy.

If phenol coefficient is more than one, the new chemical agent is more effective than phenol.

Derivatives of phenol:

. Cresols e.g. Lysol, Creolin

. Halogenated diphenyl compounds eg. Hexachlorophene It is more active on Gram-positive bacteria.

It is germicidal and anti-perspirant.

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Organic solvents

Alcohol e.g. Ethyl alcohol, Isopropyl alcohol

. Disorganize cell membrane lipid structure.

. Denatures protein.

. Active against Gram-positive bacteria, Gram-negative bacteria and acid-fast bacilli.

Uses:

1.Potent skin disinfectants

2.Disinfects clinical thermometer

NB: Ethanol is potent at concentration of 70%.

Chemical agents that denature proteins

E.g. Acids and alkalies, Quates, Alcohol

. Causes conformational alteration of proteins (unfolding of polypeptide chain) resulting in irregular looping and coiling of polypeptide chain.

Acids like benzoic acid, citric acid and acetic acid are helpful as food preservatives: extending storage life of food products.

Chemical agents that modify functional groups of proteins and nucleic acids

Heavy metals

1.Mercurials : mercuric chloride – limited use because of toxicity. Organic mercurials – less toxic than inorganic mercuric salts.

Used as antiseptics.

E.g. Merthiolate

Mercurochrome

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2. Silver compounds

E.g. Silver nitrate, Silver salfasalazine

Used as ophthalmic and wound (e.g. In burn patients) antiseptic.

Oxidizing agents

Converts functional –SH group into non-functional –S-S group.

1.Halogens e.g. Chlorine, Iodine

a. Chlorine: inactivated by organic materials.

Preparations and uses:

•Chlorine: water disinfectant; the dosage is 0.5-1.0 PPM as a disinfectant.

•Hypochlorite: sanitizing dairy and food processing industries, house holds and hospitals.

•Organic or inorganic chloramine : effective water disinfectant acting by liberating chlorine.

b. Iodine: effective skin disinfectant

Preparations:

. Aqueous Iodine

. Iodine Tincture: 2% iodine and 70% ethanol.

. Iodiphores (e.g. Betadine): Less toxic and less active than Aqueous iodine and iodine tincture.

2.Hydrogen peroxide (3%)

Used for cleansing of wound, disinfecting medical-surgical devices and plastic contact lenses.

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Dyes

E.g. Brilliant green Malachite green Crystal violet

. highly selective for Gram-positive bacteria. Uses

. for treatment of dermatological lesions.

. for formulation of selective culture media.

Alkylating agents

E.g. Formaldehydde

Glutaraldehyde

Ethylene oxide

Formaldehyde

37% aqueous solution form is named as formalin. Uses:

. Preservation of fresh tissues.

. Preparation of vaccines from bacterial surfaces, viruses and toxins.

. Bactericidal including spores.

Glutaraldehyde

. 10 times more effective than formaldehyde.

. cold sterling for medical-surgical instruments.

Ethylene oxide

. gaseous sterling chemical.

Use: sterilize medical-surgical devices that would be damaged by heat.

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Antiseptic agents: Disinfectants that are applied on animate bodies.

Characteristics:

. Never be toxic to cells

. Never be corrosive

. Should never change nature of skin Eg. Savlon

Alcohol( 70%) Iodine tincture Iodophor

1.9.2. Physical methods of sterilization and disinfection

1.Heat: the most reliable and universally applicable method of sterilization.

Mechanism of action

. Dry heat – denatures protein.

. Moist heat – denatures and coagulates protein.

1.1.Dry heat : It is less efficient and requires high temperature and long period heating than moist heat.

Dry heat can be used by the following methods:

a.Incineration : It is an efficient method of sterilization and disposal of contaminated needles, syringes and cover slips at high temperature

b.Red heat : Inoculating wires, loops and points of forceps are sterilized by holding them in the flame of a Bunsen burner until they are red hot.

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c.Flaming: Scalpels and neck of flasks, bottles and tubes are exposed for a few seconds, but it is of uncertain efficacy.

d.Hot Air Sterilizer (Oven): it is essential that hot air should circulate between the objects being sterilized and these must be loosely packed and adequate air space to ensure optimum heat transfer.

It is done by applying 160 0c for 1 hour. Use: Sterilizes glassware, oils, greases, lubricants and powders.

1.2.Moist heat: It is preferred to dry heat due to more rapid killing. Moist heat can be used by the following methods.

a.Boiling: It is not reliable method of sterilization. It is done by applying 100 0c for 30 minutes.

Used for sterilizing catheters, dressing and fabrics.

b.Tyndallization : Intermittent steaming (Fractional sterilization) Steaming of the material is done at 100 0c for 30 minutes on three consecutive days.

The principle is that spores which survived the heating process would germinate before the next thermal exposure and then would be killed. It is used for sterilizing heat sensitive culture media containing materials such as carbohydrates, egg or serum.

c.Pasteurization: It is the process of application of heat at temperature of

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62 0c for 30 minutes(Holder method) or 72 0c for 15 seconds (Flash method) followed by rapid cooling to discourage bacterial growth.

Uses:

. Pasteurization of milk

. Preparation of bacterial vaccines.

d. Autoclaving : Steam under pressure

It is based on the principle that when water is boiled at increased pressure, hot saturated steam will be formed which penetrates and gives up its latent heat when it condenses on cooler objects.

Hot saturated steam in autoclaving acts as an excellent agent for sterilization because of:

1.high temperature

2.High latent heat

3.ability to form water of condensation

4.contraction in volume that occurs during condensation

Method of using an autoclave

ƒAdd the correct volume of water to the autoclave.

ƒPlace the bottles and tubes of culture media with caps loosened in the inner chamber of the autoclave.

ƒSecure the lid i.e. Open the air-outlet and close the draw-off knob.

ƒAdjust the safety valve to the required pressure and temperature.

ƒWhen the required pressure and temperature has been reached, begin timing.

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NB: Most culture media are sterilized at a pressure of 15 lb/in2, at a temperature of 121 OC for 15 minutes.

ƒAt the end of sterilizing time, turn off the heat and allow the autoclave to cool naturally.

It destroys bacterial endospores and vegetative cells.

Uses: Sterilize solid and fluid culture media, gowns, medical and surgical equipment.

Time –Temperature-Pressure level relationship in moist heat sterilization (autoclaving)

Temperature	Time	Pressure level
121 0c	15 minutes	15 lb/inch2
126 0c	10 minutes	20 lb/inch2
134 0c	3 minutes	30 lb/inch2

Methods of controlling sterilization

1.Recording of temperature and time of each sterilizing cycle.

2.Heat-sensitive autoclave tape fixed to the outside of each

pack.

. Color change of autoclave tape from blue to brown-black indicates completesterilization.

3.Biological indicator : Use of paper strips impregnated with spores of Bacillus stereothermophilus

. Put the paper strip in the culture medium after autoclaving and observe for germinating bacteria to check for growth. In complete sterilization there should not be bacterial growth.

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e. Freezing: Inactivation of living bacteria by cold.

It prevents active multiplication of bacteria by decreasing the metabolic activity of bacteria.

Lyophilization : Freeze-drying : Involves rapid freezing with subsequent drying.

Use:

. Preservation of microbial cultures.

. Preservation of vaccines.

f.Filtration : Mechanical sieving through membrane filters.

Uses:

. Sterilization of thermolabile parental and ophthalmic solutions, sera and plasma.

. Microbial evaluation of water purity.

. Viable counting procedures.

. Determination of viral particle size g. Radiation : Ioning and ultra violet radiation

Ioning radiation includes χ ray, γ ray and β ray. These induce break down of single stranded or sometimes double stranded DNA.

Ultra violet radiation has less quantum energy with low penetrating power than ionic radiation.

Spore forming bacteria are more resistant to ionic and ultra violet radiation than vegetative bacteria because of:

1.The spore coat confers protection.

2.DNA is in different state in spores.

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Use: Sterilize surgical sutures, catheters, petridishes, culture media while dispensing and pharmaceutical products like hormones, enzymes and antibiotics.

It also sterilize biological safety cabinet (Laboratory rooms).

1.9.3. Anti-Microbial agents and Sensitivity Testing

Anti- Microbial drugs

Anti-microbial drugs include

. Antibiotics

. Chemical anti-microbials

Antibiotics:

Definition: Antimicrobial substances produced by living micro- organisms.

Chemical anti-microbials

Definition: synthetically produced anti-micorbial compounds. Anti-microbial drugs show specific toxicity to microbial cells due to differences in cell envelope, protein and enzymes to host cells.

Mechanism of action of anti-microbial drugs

1.Those inhibiting cell wall synthesis, leading to cell lyses.

-penicillin

-cephalosporin

-vancomycin

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2.Those damaging cell membrane leading to loss of cell contents and then cell death.

-polymyxin

-Amphotericin B

3.Those inhibiting protein synthesis and then arresting bacterial growth

-aminoglycosides

-tetracycline

-erythromycin

-chloramphenicol

-clindamycin

4.Those inhibiting nucleic acid synthesis 4.1. Preventing DNA synthesis

-Nalidixic acid

-Quinolones

4.2. Preventing RNA synthesis

-rifampicin

5.Those inhibiting nucleotide synthesis

-sulfonamide

-trimethoprim

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Resistance of bacteria to anti-microbial drugs

Production of enzymes that destroy or inactivate anti-microbials Eg. B.lactamase destroying B-lactam ring of penicillin

1.Altering permeability of bacterial cell membrane Eg. resistance to polymixin and tetracycline

2.Developing an altered structural target for the drug Eg. resistance to aminoglycosides and erythromycin

3.Developing an altered metabolic pathway that bypasses the reaction inhibited by the drugs

Eg. resistance to sulfonamides

4.Developing an altered enzyme that can still perform its metabolic function but is much less affected by the drug

Eg. resistance to trimethoprim

Dangers of indiscriminate use of antimicorbial drugs

1.Wide spread sensitization resulting in hypersensitivity and anaphylactic reaction, and drug rashes.

2.Changing normal microbial flora leading to “super infection” due to over growth of drug-resistant micro-organism.

3.Direct drug toxicity

Eg. renal and auditory nerve damage due to aminoglylosides toxicity.

4.Masking serious infection with out eradicating it.

5.Development of drug resistance by micro-organisms.

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Anti-microbial sensitivity testing

Anti-microbine activity is measured in vitro in order to determine:

-the potency of an anti-microbial agent

-concentration of anti-microbial agent in body tissues or fluids

-the sensitivity of a given micro-organism to known concentrations of the drug

Measurement of anti-microbial activity

Techniques

-a diffusion technique

-a dilution technique

Diffusion Sensitivity Tests

It is the routinely used sensitivity test by most microbiology laboratories.

A filter paper disk containing measured quantities of drug is placed on a solid medium that has been seeded with the test organisms. The drug diffuses from the disk into the medium.

Following over right incubation, the diameter of the clear zone of inhibition surrounding the deposit of drug is taken as a measure of the inhibitory power of the drug against the particular test organism.

Bacterial strains sensitive to the drug are inhibited at a distance from the disk whereas resistant strains grow up to the edge of the disk.

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In the kirby-Bauer technique, the zone of inhibition is measured and compared to a prepared scale, which correlates the zone of inhibition size with the minimum inhibition concentration (MIC).

NB: Minimal inhibitory concentration (MIC) is the lowest concentration of antimicrobial agent that is required to inhibit in vitro bacterial multiplication under specified conditions.

Minimal bactericidal concentration (MBC) is the least concentration of the anti-microbial required producing a sterile culture.

Dilution Sensitivity Tests

-Agar dilution tests

-Broth dilution tests

Graded amounts of antimcorbial agents are incorporated into liquid or solid bacteriology media. The media are subsequently inoculated with test bacteria and incubated.

The end point is taken as that amount of antimicobial agent required to inhibit the growth of the test bacteria (MIC) or to kill the test bacteria (MBC).

Nowadays the above tests are either time consuming or cumbersome, so the advent of microdilution both solution tests has simplified the method and permit a quantitative result to be reported, indicating the amount of a given drug necessary to inhibit or kill the test micro-organism.

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Factors affecting anti-microbial activity in vitro

1.PH of the environment

-some drugs more active at acidic or alkaline PH

2.Components of medium

-media composition components enhance or inhibit bacterial growth.

3.Stability of drug

4.Size of inoculum: the larger the bacterial inoculum, the lower the apparent sensitivity of the organisms.

5.Length of incubation: short exposure of moisture to the drug inhibits their growth but does not kill them; longer exposure of moisture to a drug gives a chance for resistant motants to emerge.

6.Metabolic activity of moistures

-actively and rapidly growing micro-organisms are more susceptible to drug action than those in the resting phase.

Techniques of routinely used antimicrobial sensitivity testing (disc diffusion tests)

Required:

-sensitivity testing media

-Anti-microbial discs

-Control strains

-Turbidity standard

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Sensitivity testing media: The commonly used media is Mueller- Hinton agar.

For pathogens requiring enriched media like Neisseria gonorrhea, Heamophilus influenzae and Streptococcus pneumoniae, it is necessary to add blood to (heat it if needed) sensitivity testing agar.

Turbidity (Opacity) standard: This is a barium chloride standard against which the turbidity of the test inocula can be compared. The turbidity of the standard is equivalent to the turbidity of subcultured broth test micro-organism.

Method

-Emulsify several colonies of similar appearance of the test organism in a small volume of sterile nutrient both.

-Match the turbidity of the subculture against the turbidity standard.

-Apply a loopful of the test organism subculture to the sensitivity testing plate using a sterile loop.

-Spread the inoculum evenly across the plate using a sterile dry cotton wool swab.

-Allow the inocula to dry for a few minutes with the petridish lid in place.

-Place the anti microbial discs into the test organism in petridish using a sterile forceps or dispenser.

NB: each disc should be pressed down on the medium and should not be moved once in place.

-Incubate the plate aerobically at 35-37 oc over night after 30 minutes of applying the discs.

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-Read the tests and interpret as ‘sensitive (S)’, “ resistant

(R)”or “ intermediate (I)” comparing the chart of the sensitivity test.

Fig. 1.16 Antimicrobial sensitivity test Media

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Review Questions

1.Mention the function of bacterial cell envelope

2.Mention the procedure of gram’s and Ziehl-Neelson’s staining method

3.Discuss the different types of culture media

4.Label and describe each bacterial growth phase

5.Describe the types of gene transfer that alter the bacterial genome

6.List and describe factors that influence antimicrobial sensitivity testing in vitro

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CHAPTER TWO

COLLECTION, TRANSPORT AND

EXAMINATION OF SPECIMEN

♦If pathogens are to be isolated successfully, the type of specimen, its collection,time and method of its dispatch to the laboratorty must be correct.

♦Adequate information about the patient’s condition and antimicrobial treatment must also be sent in the Specimen.

Type of specimen

The correct type of specimen to be collected will depend on the pathogens to be isolated.

For example: a cervical not a vaginal swab is required for the most successful isolation of N.gonorrhoeae from a woman.

:sputum not a saliva is essential for the isolation of respiratory pathogens.

Time of collection

™Specimens such as urine and sputum are best collected soon after a patient wakes when organisms have had the opportunity to multiply over several hours.

™Blood for culture is usually best collected when a patient’s temperature begins to rise.

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™The time of collection for most other speciemens will depend on the condition of the patient, and the time agreed between medical, nursing and laboratory personnel for the delivery of the specimen to the laboratory.

™Every effort must be made to collect specimens for

	microbiological investigation before antimicrobial treatment is
	started.
Collection techniques
♦	The laboratory should issue written instruction to all those
	responsible for collecting specimens including staff of wards,
	out patient clinics and health centres.
	♦ Precaution apply to the collection of most microbiological
	specimens:
	1) Use	a collection technicnque that will ensure a
	specimencontains only those organisms from the
	site where it was collected.
	•	If contaminating organisms are introduced in
		to a specimen during its collection or
		subsequent handling, this may lead to
		difficulties in the interpretation of cultures and
		delays in the issuing reports.
	•	A strictly sterile (aseptic) procedure is
		essential when collecting from site that are
		normally sterile.

Example: collection of blood, CSF or effusions

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This teachings is necessary not only to prevent contamination of the specimen but also protect the patient.

2)Avoid contaminating discharges or ulcer material with the skin commensals. The swabs used to collect the specimens must be sterile and the absorbent cotton wool from which the swabs are made must be free from antibacterial substances.

3)Collect specimens in sterile, leak- proof, dry containers, free from all traces of disinfectant.

Container must be clean but need not be sterile for the collection of feaces and sputum.

4)Those responsible for collecting specimens should report any abnormal features, such as coldiness in a specimen which should

appear clear, abnormal coloration or presence of pus, blood, mucus or parasites.

Labelling of specimens and sending of a request form

Each specimen must be accompanied by a request form which gives:

-The patient’s name, age (whether an infant, child or adult), number, and ward or health center.

-Type of specimen and the date and time of it collection.

-Investigation required

-Clinical note giving details of the patient’s illness, suspected disease, and any antimicrobial treatment that may have been started at home or in the hospital.

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Specimen containing dangerous pathogen

™Those delivering, receiving and examining specimens must be informed if a specimen is likely to contain highly infection organisms. Such specimen should be labelled HIGH RISK, and whenever possible carry a warning symbols such as red dot, star or triangle

™Specimen which should be marked as HIGH RISK include:

•Sputum likely to contain M. tuberculosis

•Faecal specimen that may contain V. cholerae or S.typhi

•Fluid from ulcers postules that may contain anthrax bacilli or treponemes

•Specimens from patient with suspected HIV infection, hepatitis, viral haemorrhagicfever, or plague.

Preservatives and transport media for microbiological specimen

™In general, specimens for microbiological investigations should be delivered to the laboratory as soon as possible. This will help to ensure that pathogens are living when they reach the labratory.

™When a delay in delivery is unavoidable,

For example, when transporting a specimen from health center to a hospital laboratory, a suitable chemical preservative or transport culture medium must be used.

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™This will help to prevent organisms from dying due to enzyme action, change of pH, or lack of essential nutrients.

Example. Amies transport medium widely used and effective in ensuring the survival of pathogens like the more delicate organisms such as Neisseria gonorrhoeae.

Cary - Blair medium in used as transport medium for faeces that may contain Salmonella, shigella, campylobacter or vibro species.

Example of Preservatives include boric acid added to urine and cetylpyridinium- chloride sodium chloride (CPC-Nacl) added to sputum for the isolation of M.tuberculosis.

Transport of microbiological specimens collected in a hospital

•Specimen should reach to the laboratory as soon as possible or a suitable preservative or transport medium must be used.

•Refrigeration at 4-10 0c can help to preserve cells and reduce the multiplication of commensals in unpreserved specimens.

•However, specimens for isolation of Haemophilus, S. pneumoniae or Neisseria species, must never be refrigerated because cold kills these pathogens.

™If specimen are to be mailed, the regulations regarding the sending of “pathological specimens” through the post should be obtained from the post office and followed exactly. When dispatching microbilogical speciemens:

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1)Keep a register of all specimens dispatched. Record Name

Number

Ward or health centre of the patient. Type of specimen

Investigation required Date of dispatch

Method of sending the specimen (eg. Mailing, hand –delivery etc.)

2)Ensure that the specimen container is free from cracks, and the cap is leak proof. Seal round the container cap with adhesive tape to prevent loosening and leakage during transit.

3)If the container is glass tube or bottle, use sufficient packaging material to prefect a specimen.

If the specimen is fluid, use sufficient absorbent materiel to absorb it.

4)Mark all specimen that may contain highly infectious organism “HIGH RISK”

5)Dispatch slides in a plastic slide continer or use slide carrying box.

6)Label speciments dispetched by mail, “FRAGILE WITH CARE- PATHOLOGICAL SPECIMGN”.

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Collection, Transport and examination of sputum

Possible pathogens
Gram positive	GRAM NEGATIVE
Streptococcus pnemoniae	Haemophilus influenzae
Staphylococcus aureus	Klebsiella pneumoniae
Streptococcus pyogenes	Pseudomonas aerugnosa
	Proteus species
	Yersina pestis

Sputum commensals

™Sputum as it is being collected passes through the pharynx and the mouth. Therefore, it becomes contaminated in the small number of commensal organisms from the upper respiratory tract and mouth

Gram positive	Gram negative
Staphyloccus aureus	Neisseria
Staphyloccus epidermidis	Branhamella catarrhac’s
Streptococcus Viridans	Haemophilus influenzae
Streptococcus pnemoniae	Fusobacteria
Enterocci	Coliforms
Diphtheroids
Yeast-lke fungi

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In a hospital with a microbiology Laboratory.

1.Give the patient a clean (need not be sterile), dry, wide- necked, leak- proof container and request him or her to cough deeply to produce a sputum specimen

Note: The specimen must be sputum, not saliva.

Sputum is best collected in the morning, soon after the patent wakes and before any mouth-wash is used.

If the patient is young child and it is not possible to obtain sputum, gastric washings can be used for the isolation of M.tuberculosis but not for other respiratory

2.Label the container and fill the request form.

3.If pneumonia or bronchopneumonia is suspected, deliver the sputum to the laboratory with as little delay as possible.

•Because organisms such as S.pneumoniae and H. influenza require culturing as soon as possible.

•If the specimen is for the isolation of M.tuberculosis, ensure it is delivered to the laboratory within 2 hours or kept at 4 Oc until delivery is possible.

•If pneumonia plague is suspected: Deliver the sputum to the laboratory as soon as possible. Make sure that the specimen is marked HIGH RISK.

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For dispatching to a microbiology laboratory

Collect the sputum in a container as usual.

Depending on whether the sputum is for the isolation of pneumonia and bronchopneumonia or M.tuberculosis, proceed as follows:

Pneumonia and Bronchopneumonia pathogen

™Collect a purulent part of the sputum on a cotton wool swab, and insert in a container of Amies transport medium. Label the container using a lead pencil

™Amies will help the pathogens to survive and from being overgrown by fast-multiplying commensas.

™Make a smear on slides for gram staining and fix using heat or alcohol and send the swab and the request form to reach to the laboratory.

M.tuberculosis

•Make a smear of the sputum on slides for Ziel- Neelsen staining, from the most pumlent materials.

•Fix with alcohol.

If pneumonia plague is suspected:

-Send the swab of the sputum in cary-Blair transport medium to reach the microbiology laboratory.

Caution: Y. pestis is highly infectious organism.

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Laboratory examination of sputum

1.Describe the appearance of the specimens

•Purulent: Green- looking with pus and mucus.

•Mucopurulent: Green- looking with pus and Mucus

•Mucoid: Mostly mucous

•Mucosalivary: Mucus with a small amount of saliva

•Bloody: should be reported.

2.Examine the specimen microscopically.

Gram smear

Look for pus cells and bacteria

-Gram positiveve diplococci that could be S. pneumoniae

-Gram positive cocci that could be S. aureus

-Gram negative rods that could be H. influenzae

-Gram negative capsulated rods that could be K. pneumoniae.

Additional investigation

Look for:

-saline preparation, if paragonimiasis is suspected

-Eosin paration, if asthma or other allergic condition is suspected.

-Potassium hydroxide (KoH) preparation, if fungal infection is suspected (yeast cells, Nocardia species, actinomycetes).

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-Giemsa smear, if histoplasmosis or pneumonic plague is suspected.

3)Culture the specimen

To obtain as pure a culture as possible of a respiratory pathogen it is necessary to reduce the number of commensals inoculated.

Ways of reducig commensal numbers include washing the sputum free from saliva or liquefying and diluting it.

Blood agar and chocolate agar

-Wash a purulent part of the sputum in about sml of sterile physiological saline.

-Inoculate the washed sputum on plates of blood agar and chocolate (heated blood) agar.

-Add an optochin disc to the chocolate agar plate. This will help to identity S. pneumonia.

-Incubate the blood agar plate aerobically and the chocolate agar in a co2 enriched atmosphere at 35-370c for upto 48 hours, examining for growth atfter ovemight incubation.

Additional:

Lowenstein Jensen medium, if pulmonary tubercles is suspected.

-About 20 minuets before culturing, decontaminate the specimen by mixing equal volumes of sputum and

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sodium hydroxide (NaoH) 40 g/l or ( 4% W/v ) solution. Shake at intervals to homogenize the sputum.

-Using a sterile pasteur pepitte, inoculate 200 Nl (0.2ml) of the well-mixed homogenized sputum on a slope of

acid Lowenstein Jensen medium. Allow the specimen to run down the slope.

-Incubate at 350c –370c in a rack placed at an agle of about 450c to ensure that the specimen is incontact with the full length of the slops.

-After one week, place the slope in an upright position and continue to incubate, examining twice a week for groth.

4.Examine and report the culture

Examine the blood agar & Chocolate agar culture for:

S.pneumonia S. aureus H. influezae K. pneumoniae Pse. Aeruginosa

Examin Lowenstein Jensen e culture for:

M..TUBERCULOSIS

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Collection, transport and examination of mouth and throat specimens

Possible pathogens
Gram positive	Gram negative
Streptococcus pyogenes	Vincent’s organisms
Other beta-haemolytic streptococci
Corynebacterium diphtheriae

Virus: Respiratory viruses, enteroviruses and herpes simplex virus type-I

Fungi: candida albicans.

•Pathogens in the upper respiratory tract such as Bordetella, pertussis, streptoccus pneumoniae, and Neisseria meningitidis, are usually more successfully isolated from pernasal and nasopharyngeal specimens.

Throat and moth commensals

Gram positive	Gram negative
Viridans streptococci	Branhamella catarrhalis
Non-haemolytic streptococci	Neisseria Pharyngitidis
Streptococcus pneumonia	Fusobacteria
Staphylococcus epidermidis	Coliform
Micrococci
Lactobacilil
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Collection and Transport of Throat and Mouth swabs

•Whenever possible it should be collected by a medical officer or experienced nurse.

In a hospital with a microbiology laboratory

1. In a good light and using the handle of a spoon to depress the tongue, examine the inside of the month.

Look for inflammation, and the presence of any membrane, exudate or pus.

•With diphtheria – a greyish- yellow membrane (later

becoming grayish Green-black and smelly) can often seen over the soft palate and backwards on to the phargedl wall.

•With streptococcal sore throat, the tonsil are inflammed and often coverd in a yellow spots.

•With infectious mononucleosis, the tonsils may be covered with a white exudate

•With C. albicans infection, patches of white exudate may be seen attached in places to the mucosal membrane of the month.

2.Swab the affected area using a sterile cotton. Taking care not to contaminate the swab with saliva, return it to its sterile container.

•Before swabbing the patient must not treated with antiboitics or antiseptic month-washesr (gargles) for 8 hours.

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•For children, swabbing may causes obstruction child’s airway instead blood for culture should be collected.

3.Within 2 hours of collection, deliver the swab with a request form to the laboratory

Dispatching to the microbiology laboratory

1.Using a sterile swab or silica gel collect a specimen from the infected area.

2.Taking care not to contaminate the swab, return it to its tube.

Seal with adhesive tape and label using a lead pencil.

3.Send the swab with its request form to reach the microbiology laboratory within three days.

Laboratory Examination of throat and month swabs

1.culture the specimen Blood agar

-Inoculate the swab on a plate of blood agar.

-If the swab is received in silica gel ( eg. from health centre), moisten it first with sterile nutrient broth and then inoculate the plate.

-Add a bacitracin disc. This will help in the identification of S. pyogenen.

-Incubate the plate preferably anaerobically or in a co2 enriched atmosphere overnight at 35-370c

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•Beta-haemolytic streptococci produce larger Zones of haemolysis when incubated anaerobically.

-A minority of Group A streptococcus strains will only grow if incubated anaerobically.

Additional

Modified Tinsdale medium and tellurite blood agar if diphtheria is suspected.

•If in an area where diphtheria occurs or the disease is suspected, inoculate the swab on modified Tinsdale medium and tellurite blood agar.

•Incubate the plates aerobically at 35-370c for up to 48 hours, examing for growth after overnight incubation.

Sabouraud agar if thrush is suspected

•Inoculate the swab on sabourad agar

•Incubate at 35-370c for up to 48hours checking for growth after overnight incubation

2.Examine the specimen microscopically

Gram smear

Examine the smear for pus cells and Vincent’s organisms:

•Vincent’s organism are seen as Gram negative spirochaetes ( B. vincenti)

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And fusiform rods, (L. buccalis).

•If thrush is suspected, look for Gram positivee yeast-like cells.

•If diphtheria is suspected, look for Gram positive pleomorphic rods

•Commensal diphtheroids, however, are strongly Gram

positive and Unlike

C.diphtheriae, they show little variation in size or shape.

Additional

Albert stained smear

Examine the smear for bacteria that that could be C. diphtheriae:

•Most strains of C. diphtheriae contain dark-staining volutin gtanules.

•The pleomorphic rods tend to join together at angles giving the appearance of Chinese letters.

3.Examine and report the cultures.

Blood agarculture

•look for beta-haemolytic colonies that could be group

Astreptococcu(S. pyogenes)

or a beta-haemolytic streptococcus belonging to another

lancefield group

such as group C or G.

•Most group A strains show sensitivity to bacitacin.

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Reporting of the throat swab cultures:

•If a B-haemolytic streptococcus sensitive to bacitracin is isolated, report the culture as “S. pyogenes presumptive group A isolated, Lancefield group to be confirmed.

•If a B-haemolytic streptococcus that is not sensitive to bacitracin is isolated (confirm that the colonies are

streptococci), report the culture as ”Beta-haemolytic streptococcus isolated, lancfield group to follow”.

Additional:

Modified Tinsdale medium (MTM) and tellurite blood agar (TBA) Cultures.

•On MTM, c. diphtheriae produces grey-black raised colonies surrounded by a dark brown area.

-If there is no growth, reincubate to the plate for further 24 hours.

•Examine the TBA plate for grey or grey-black colonies measuring 0.5-2 mm in diameter.

Sabouraud agar culture Look for candid albicans

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Collection transport and examination of Nasopharyngeal aspirates and Nasal swabs

Nasopharyngeal Aspirates and perinasal swabs

Possible pethogens
Grampositive	Gram negative
Streptococcus pneumonia	Haemophylus influenzae
Corynebacterium diphtheriae	Neisseria meningitidis
	(carriers)
	Bordetella pertussis
	Bordetella parapertussis
	Klebsiella species

Also M. leprae

Viruses: Respiratory viruses and entrovruses.

Anterior Nasal Swabs

Possible pathogens

•Most anterior nasal swabs are examined to detect carriers of pathogens

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Gram positive	Gram negative
S. aureus	N. meningitidis
S.pyogenes	H. influenzae (mostly non capsulate)

Nasopharyngeal Aspirates, perinasal and Anterior Nasal swabs Commensals

Gram positive	Gram negative
S. viridans	Neisseria species
S. pneumoniae	Haemophilus species
Entrococci
Staphylococcus species
Micrococci

Collection and Transport of upper Respiratory Tract specimens

Nasopharyngeal aspirates: Gently pass a sterile catheter through one nostril as far as the nosopharynx.

:Attach a sterile syringe to the catheter, and aspirate a specimen of mucopus.

:Dispense the specimen into a small sterile container.

:Label and deliver with a completed request form to the laboratory as soon as possible.

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Pernasal swabs for the culture of B. pertussis:

If whooping cough is suspected and the nasal passages are clear, collect a penasal swab as follows:

1. Using a sterile cotton or alginate wool swab attached to an easily bent pieces of wire, gently pass the swab along the floor of one nostril directing the swabdown wards and backward as far as the Nasopharynx.

2.Taking care not to contaminate the swab, replace it in its sterile container.

3.Label, and deliver immediately to the laboratory

with a request form.

Note:- B. pertusis does not survive well on a swab -It must be cultured as soon as possible.

-If plating cannot be performed at the bedside, the swab should be placed in special transport medium

-If it is not possible to collect a per nasal swab, a less satisfactory way of isolating B. pertussis is to hold the plate of culture medium in front of the child’s month during a coughing attack.

Anterior nasal swabs to detect carriers:

1.Using a steile cotton wool swab moistened with sterile peptone water, gently swab the inside surface of the nose.

2.Taking care not to contaminate the swab, replace it in its sterile container.

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3.Label, and within 2 hours deliver the swab with a request form to the laboratory.

Laboratory examination of upper respiratory tract specimen

1.culture the specimen Blood agar and chocolate agar

•To detect H. influenzae, N. meningitidis, and S. aureus carrier:

-Inoculate the swab on chocolate (heated blood) agar.

-Incubate the plate in carbondioxide enriched atmosphere at at 350 –370c for up to 48 hours., examinig for growth after overnight incubation.

•To detect S. pyogenes and S. aureus carriers:

-Inoculate the swab on blood agar.

-Incubate the plate preferably anaerobically at 350 –370c overnight (if for the isolations of S. aureus only, incubate aerobically).

Additional

Charcoal Cephalexin blood agar if whooping cough is suspected

-Inoculate the swab over the entire surface of a plate of charcoal cephalexin blood agar (CCBA).

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-Incubate the plate aerobically in a moist atmosphere

(in a plastic bag or polythene container with a wet piece of cotton wool) at 35-370c for up to 6 days,

-Examining for growth after about 48 hours incubation.

Culture of swab received in bordetella transport medium:

	- Inoculate the swab on a plate of CCBA
	- Return the swab in its container and incubated it at 35-
	370c
	- Examine the plate for growth after 48hoursrs incubation.
™	If no growth is seen, inoculate a second
	plats of CCBA with the incubated swab.

-Reincubate the first plats for a further four nights.

-Examining for growth every 24 hours.

-The second plate for up to 6 days.

2.Examine and report the cultures

Blood agar and chocolates agar cultures(routine)

Look for coloniess that could be

H. influenzae

Neissena mengitdis

S. aureus

S. pyogenes (Group A)

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Additional

Charcoal ceplea lecin blood agar (CCBA) culture

The examination of a CCBA culture for berdetela species and the identification of B. pertussis and B. parapertussis.

Collection, Transport and examination of Ear Discharges

Possible pathogens

Gram positive	Gram negative
S. aureus		P. aeruginossa
S.pyogenes		H. influenzae
Other beta-haemolytic streptococci		Klebsiella specia
S. preumoniae		proteus species

E.coli and other coliforms

Bacteriodes species

Fungi: Aspergillus species especially A. niger, candida species, and occasionally various species of dermatophyte or phycomycete.

A fungal infection of the ear is called otomycosis

™External Ear infection are more commonly caused by:

S.aureus

S. pyogenes P. aeruginos.

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Middle ear infection or otitis media are commonlycaused by :

s.pneumoniae s. pyogenes

and other B- haemolytic streptococci H.influenzae(esp. in children) coliforms

Klebsiella spenil S. aureus

P. aeruginosa

™Chronic ear infection are often caused by Bacteroides species or fungi

Commensals

The middle ear is normally sterile. The following organisms may be found as commensals in the external ear:

Gram positive	Gram negative
Viridans streptococci	Escherichia coli and
other coliforms S.epidermidis
Bacillus species
Corynebacterium species

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Collection and Transport of Ear Discharges

1.Collect a specimen of the discharge on a sterile cotton.

2.Place it in container of Amies transport medium, breaking off the swab stick to allow the bottle top to be replaced tightly.

3.Make a smear of the discharge on a slide (for Gram staining).

4.Label the specimens and send them with its request form to the laboratory Within 6 hours.

Laboratory examination of Ear Discharges

1. culture the specimen

Blood agar and Macconkey agar

Inoculate the specimen on blood agar and macconkeg agar Incubate both plates aerobically at 35-370c overnight.

Additional: Chocolate agar if the patient is a child: Inoculate the specimen on chocolate (heated blood) agar for the isolation of H. influenza.

Incubate the plate in a carbon dioxide enriched atmosphere at 35-370c for up to 48 hours, examining for growth after overnight incubation.

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Blood agar (Kanamycin) for anaerobic incubation if the infection is chronic

Inoculate the specimen on blood agar, preferably that which contains Kanamycin to inhibit the growth of commensals.

Incubate the plate anerobically for up to 48hours, checking for growth after overnight incubation.

Sabouraud agar if a fungal infection is suspected

Inoculate the specimen on sabouraud agar, and incubate at room tempreture for up to 6 days.

2.Examine the specimen Microscopically

Gram smear

-Make an evenly spread of the specimen on a shide.

-Allow the smear to air-dry in a safe place.

-Fix and stain by Gram technique.

-Look for pus cells and bacteria

•Gram positive cocci that could be S.aureus

•Gram positive streptococci or diplococi that could by streptococci pathogens

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•Gram negative rods that could be H.influenzae, p. aeroginosa, klebsiella specis, proteus species, E.coli or other s.

•Gram positive yeast cells that could be candida species.

•Small numbers of Gram positive cocci, streptococci, rods and also Gram negative rods may be seen in smears of ear discharges because these organisms form part of the normal microbial flora of the external ear.

Additional:

Potassium hydroxide preparation if a fungal infection is suspected

-Mix a small amount of the specimen with a drop of potassium hydroxide, 200g/l (20%W/v) on a slide, and cover with a coverglass.

-After 10 minutes, or when the preparation has cleared sufficiently, examine microscopically using 10x or 40x objective.

Look for:

•Brnaching septate hyphae with small round spores, that could be Aspergillns speies

•Pseudohyphae with yeast cells, that could be candida specis (Gram positive)

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•Branching septate hyphae, that could be a species of der matophyte

•Branching aseptate hypae, that could be a species of phycomycete.

3.Examine and Report the culture

Blood agarand MacConkey	Examine chocolate agar
S. aureus	H. influenza
B-haemolytic streptococcus
E.Coli
Profeus spese
P. aeruginosa
S. pneumonia
examine sabouroud agar for:
examine the anaerobic blood	fungi elements
agar mete for:
Bacteriode species

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Collection, transport and examination of eye specimens Possible pathogens

Gram positive	Gram negative
Staphylococcus aureus	Nesseria gonorrhoeae
Streptococcus pneumonae	Haemophilus influenzae
Streptococcus agalactiae (Group – B)	Haemophilus aegyptius
Streptococcus Pyogenes (Group – A)	Pseudomonas aeroginosa
Other B- hemolytic streptococci	Moraxella lacunata
	Erterobacteria

Chlamydiae: C trachomatis

Viruses: Adenoviruse, herpesviruses and enteroviruses

Note:

1.Inflammation of the the delicate membrane lining the eyelid and covering the eyeball conjunctiva is called conjunctivitis.Bacterial conjunctivitis is mainly caused by S. aureus, H. influenzae, H.aegyptius, S. Pneumoniae and occasionally by Moraxella lacunata.

2.Eye infection of new born infants can be caused by:

ƒN. gonorrhoeae, transmitted as the infant passes down the birth canal. It causes a severe purulent conjunctivitis that can lead to blindness if not treated. Infection usualy develops within 72hours of birth.

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ƒStreptococcus Group B (S.agalactiae) and other B-hemolytic streptococci that can be transmitted during birth.

ƒC. trachomatis, that is transmitted during birth and causes conjuctivitis 5-12 days after birth.

ƒS. aureus that is acquired after birth (commonly referred to as “sticky eyes”)

3.C. trachomatis serotype A, B and C cause endemic trachoma, a major cause of blindness especially in children and also in adults in rural areas of developing countries.

4.P. aerogunisa eye infections are frequently caused by the use of contaminated eye drops.

5.Herpes simplex virus can cause severe inflammation of the cornea (Keratitis)

Commensals

-That may be found in the eye discharges:

Gram positive	Gram negative
Viridans streptococci	Non-pathogenic neisseriae
Staphylococci	Moraxella speires

Collection and transport of eye specimen

•Eye specimen should be collected by medical officer or experienced nurses. Conjunctival scrapings to detect C. trachomatis must be collected by medical person.

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•Specimen from the eye must be cultured as soon as possible after collection because the natural secretions of the eye contain antibacterial enzymes.

Procedure

1.Using a dry sterile cotton wool swab, collect a specimen of discharge (if an inflant, swab the lower conjunctival surface).

2.Inoculate the discharge on the following culture media:

•Blood agar

•Chocolate (heated blood) agar

•MNYC N. gonorrheae selective medium, if the patient is a newborn infant.

3.Make a smear of the discharge on slide (frosted-ended) for staining by the Gram technique. If C. trachomatis infection of the newborn is suspected, make a second smear for staining by thr Giemsia technique and label the plate and slide.

4.As soon as possible, deliver the inoculated plates and smear(s) with request form to the laboratory.

Laboratory examination of the eye specimen

1.Culture the specimen Routine:

Blood agar and chocolate agar

•Inoculate the eye discharge on blood agar and chocolate (heated blood) agar.

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•Incubate the blood agar aerobically at 35-370C overnight.

•Incubate the chocolate agar plate (CAP) in a CO2 enriched atmosphere for 48hours, checking for growth after overnight incubation.

Additional

MNYC selective medium if gonococcal conjunctivtis is suspected (infant less than 3 weeks old).

•Inoculate the discharge on the plate

•Incubate at 35-370C in a CO2 enriched atmosphere overnight.

Loeffler serum slope if Moraxella infection is suspected:

•Inoculate the eye discharge on a loeffler serum slope.

•Incubate at 35-370C overnight.

2.Microscopically examination

Routine:

Gram smear

Look for:-

•Gram negative intracellular diplococci that could be N. Gonorrhoeae. If found, a presumptive diagnosis of gonococcal conjunctioitis can be made A cervical swab from the mother should also be cultured for the isolation of N.gonorrhoae.

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•Gram positive streptococci or diplococci that could be streptococci pathogens.

•G ram positive cocci that could be S.aureus

•Gram negative rods that could be Haemophilus species.

Additional

Giemsa smear if C. trachomatis infection is suspected:

1)Fix the air-dried smear by covering it with methanol for 3 minutes.

2)Dilute the Giemsa stain in the buffered water

C.trachomatis, dilute the stain 1 in 40:

-Fill asmall cylinder to the 19.5 ml (mark with buffered water)

-Add 0.5ml Giemsa stain to 20ml mark. Eg. 0.5ml + 19.5ml

For Other organisms, 1ml + 19ml

3)Stain the smear with diluted Giemsa in dish

C.trachomatis, stain 1 ½ - 2 hours

For other organisms, stain 25-30 minutes

4)Wash the slide from the dish and rise the smear with buffered water. And let the smear to air-dry and examine with oil immersion objective.

Result

9trachomatis inclusion bodies ---------- Blue-mauve to dark purple. Depending on the stage of development; If the inclusion body is more mature, it will contain ---- red- mauve stiaing elementary particles.

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9Chlamydia bodies must be differentiated from bacteria that may infect the eye such as neisseriae, Hemophilus species, staphylococci, streptococci, and moraxella species.

9All these bacteria, with the exception of Hemophilus species, stain dark blue in Giemsa stained smear, but Haemophilus rods stain pale blue.

9Report the smear as “chlamydial inclusion bodies present” or “No chlamydial inclusion bodies seen”.

Examine and Report the cultures

Blood agar and chocolate agar

Look for colonies that could be:

Haemophylus Influenzae

Staphylococcus aureus

Beta- helmolytic streptococci

Streptococcus Pneumoniae

Pseudomonas aeruginosa

Loeffler serum slope culture

Look for Moraxella lacunata

Additional

MNYC Medium culture

-	Look for N. gonorrheae
9	. oxidase positive.
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9. CHO utilization test can also confirm the organism

9. The organism should also be tested for B- lactamase production.

	Glu	lact	mal	suc
N.gonorrhoeae	A	-	-	-
N.meningtidis	A	-	A	-

Glu =glucose, Lac= lactose, mal=maltose, suc,= sucrose, A= acid produced

Acid produced (yellow or orange yellow)

Collection, transport and examination of skin specimen Possible pathogens

Gram positive	Gram negative
S. aureus	Escherichia coli
S. pyogenes	Proteus
Enterococci	Pseudomonas aeruginosa
Anaerobic streptococci	yersinia pestis
Bacillus anthracis	Vincent’s organisms
Corynebacterium ulcerans
Also M. leprae	Virus: pox viruses and herpesviviruses
M. ulcerans	Fungi: Ringworm
T. Pertenue	parasite: Leishmania spps
T. Carateum	: onchocerca volvulus
	:D. medinensis
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Commensales
Gram positive	Gram negative
Staphylococci	Escherichia Coli and other coliforms
Micrococci
Anaerobic cocci
Viridans streptococci
Enterococci
Dephtheoids

Collection of skin specimens and ulcer materials

1.Using a sterile dry cotton wool swab, collect a sample of discharge from the infected tissue.

If there is no discharge, use swabmoistened with sterile physiological saline to collect a specimen.

Insert the swab in a sterile tube.

¾If the tissue is deeply ulcerated and necrotic (full of dead cells); Aspirate a sample of infected material from the side wall of the ulcer using a sterile needle and syringe.

¾Fluid from pustules and blisters: Aspirates a specimen using a sterile needle and syringe.

¾Serous fluid from skin ulcers, papillomas or papules, that may contain treponemes:

•Collect a drop of the exudates directly on a clean cover glass and invert on a clean slide.

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•Delivery immediately the speciemen to the laboratory for examination by dark- field microscopy.

2.If the specimen has been aspirated, transport the needle and syring in a sealed water proof container immediately to the laboratory.

Laboratory examination of skin specimens

1)Culture the specimen

Blood agar and MacConkey

• Inoculate the specimen

• Incubate both plate aerobically at 35-370C overnight.

Additional:

Sabourand agar if a fungal infection is suspected

•Inoculate to agar plate

•Send to a Mycology Reference laboratory.

Modified Tinsdale Medium (MTM), if cutaneous diphitheria is suspected:

•Inoculate the sample for isolation of C. Ulcerans

•Incubate aerobically at 35-370C for up to 48hours,

examining the growth after overnight incubation.

Blood agar and MacConkey agar at room temperature, if bubonic plague is suspected:

•Inoculate the specimen

•Incubate both pletes aerobically at room temperature far up to 48hours.

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•Examination for growth after overnight incubation. 9 Y. pestis is a highly infectious organism. Maximum care should be taken

Lownstein Jensen (LJ) Meduim if Buruli ulcer is suspected:

•Decontaminate the swab by immersing it in sodium hydroxide 40g/l (4% W/v) solution for 10 minutes.

•Inoculate the decontaminated specimen on two slopes of acid LJ medium.

•Incubate one slope at 35-370C as described for culture of M. tuberculosis and incubate the other slope at 320C for up to 8 weeks.

2.) Microscopical examination

Routine:

Gram Smear:

Look for:

•Gram positive cocci that could be S. aureus

•Gram positive streptococci that could be S. pyogenes or other streptococci.

•Gram negative rods that could be p. aeroginosa, proteus speices, E.coli or other coliforms.

9 If tropical ulcer is suspected, look for vincent’s organisms

9 If cutaneus anthrax is suspected, look for large Gram variable rods lying in chains that could be B. anthracis.

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•Examine also a polychrome methylene blue

stained smear.

9If bubonic plague is suspected, look for Gram negative coccobacilli that could be Y. pestis.

Additional:

Potassium hydroxide preparation, if ringworm or other superficial fungi infection is suspected.

For detection of ringworm:

Giemsa techniques or wayson`s techniques,if bubonic plague is suspected.

Polychrome Loefler methylene blue smear if cutaneous anthrax is suspected.

-Fix with potassium permanganates 40g/l solutions for 10 minutes.

-Look for chain of large blue-stained capsules characteristic of B. anthracis.

Ziel-Neelsenstained smear if buruli ulcer is suspected

examine for acid fast bacilli.

Dark-field microscope to detect treponemes

-look for motile treponeme if yaws or pinta is suspected

Examine and report the culture

Blood agar and MacConkey agar cultures

Look for: S. aureus	Aditional investigations
S. pyogenes	sabouraud agar
P. aeniginosa	-if fungal infection is suspected
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Enterococci	Lowenstein Jensen
Proteus species	-if Buruli ulcer is suspected
Escherichia coli	MTM
	- if cutaneous diphtheria is suspected

Collection, Transport and Examination of urogenital specimens possible pathogens

Urethral swabs

•N. gonorrhoeae

•S. Pyogenes

•Ureaplasma urealyticum

•Chlamydia trachomatis and

•Occassionally Trichomonas vaginalis

Cervical swabs from non-puerperal women:

•N. gonorrhoeae

•S. pyogenes

•Other B.hemolytic streptococci

•Chalmydia trachomatis and

•herpes simplex virus

Cervical swabs from women with puerperal sepsis or septic abortion:

•S. pyogenes

•other B – haemolytic

•streptococci

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•anaerobic streptococci

•enterococci

•S. aureus

•clostridium pertfringes

•Listenia monocytogenes

•Bacterioles species

•protens species

•E. Coli & other coliform

Vaginal Swabs:

T.vaginalis candida species

Gardnerella vaginalis (Haemophilus vaginalis) Neisseria gonorrhoeae.

Fluid and pus from genital ulcers

T.pallidium C. trachomatis

Calymmatobacterium granulomatis (Donovania granulomatis) H. ducreyi.

Collection and transport of urogenital specimen

•Amies medium is the most efficient medium for transporting urethral, cervical and vaginal swabs.

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Specimen required for diagnosis of gonorrhoea

Male patients:

. Smears of urethral discharge

. Rectal swab from homosexual patient Female patients

. Smears of mucopus from the cervix and urethra

Note: N. gonorrhoeae infects the mucaus membranes of the cervix, not the vagina.

The pathogen is, therefore, more likely to be isolated from a cervical swab than from a vaginal swab.

Collection of Urethral specimens

1.cleans the urethral opening using a swab moistened with sterile physiological saline.

2.Gently massage the urethra from above downwards, and collect a sample of pus on a sterile cotton wool swab.

•The patient should not have passed urine preferably for 2hours before the specimen is collected.

3.Make a smear of the discharge on a slide for staining by the Gram technique and label the specimen.

Collection of Cervical specimen

1.Moisten a vaginal speciemen with sterile warm water and insert into the vagina.

2.Cleause the ceroix using a swab moistened with sterile physiological sahrie.

3.Pass a sterile cotton wool swab into the endocervical canal and gently rotate the swab to obtain a specimen.

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•Amies transport

•Make a smear of the cervical mucopus for Gram stainingl and lable the specimen

Collection of vaginal specimen

•Using sterile swab, collect a sample of vaginal discharge.

-Amies

-Smear and label the sample

Laboratory examination of urogenital specimen

Routine culture

•MNYC medium

Incubate in moist CO2 atmosphere

⇒Neisseria gonorroeae

Microscopy

⇒Gram smear:

Look for pus cells and bacteria

Suspected gonorrhoeae:

Look for intracellular gram negative diplococci

vaginitis:

Look for yeast cells, and epithelialcells in the gram variable coccobacili

Suspected puerperal sepsis or septic abortion

Look for gram positive rods, streptococci, cocci and gram negative rods.

Suspected chanchroid

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Look for Gram negative coccobacilli showing bipolar staining

Additional culture

Blood agar (aerobic and anaerobic), macCokey

agar,and cooked meat medium, if puerperal sepsis or septic abortion is suspected

Sabourand medium, if vaginal candidiasis is suspected and yeast cell not detected microscopically

Serum culture, if chancroid is suspected ⇒H. ducreyi

Microscopy

Sahlie preparation, if trichomoniasis is suspected. Gemsa stained smear: If donovanosis is suspected Dark field preparation, if syphilis is suspected.

Colleciton, transport and examination of cerebrospinal fluid

Possible pathogens

Gram positive

S.pneumonia S. aureus

S. agalactiae (Group B) Listeria monocytogenes Bacillus anthracis

Gram negative

Neisseria meningitides

H.influenzae type b

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Escherichia coli

Psendomonas aeraginosa

Proteus species

Salmonella species

⇒Also M. tuberculosis

Viruses:

Enteroviruses, especially echoviruses and coxsackieviruses. Rarely polioviruses may also be isolated from CSf.

Fungi: Cryptococcus neoformans

Parasites: Trypanosoma species Naegleria fowleri

Acanthamoeba species and rarely the larvae of Angiostrongylus cantonensis and Dirofilaira immitis

Note:

1.Inflammation of the meninges (membranes that cover the brain and spinal cord) is called meningitis.

Pathogens reach the meninges in the blood stream or

occasionally by spreading from nearby sites such as the middle ear or nasal sinuses.

Meningitis is described as:

•Pyogenic (purulent), when the C.S.f contains mainly poly morphonuclear neutrophils (pus cell), as in acute meningitis caused by N. meningitides, H. influenzae and S. pneumonae.

•Pus cells are also found in the C.S.f in acute amoebic meningoencephalitis.

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•Lymphocytic, when the C.S.f contains mainly lymphocytes, as in meningitis caused by viruses, M. tuberculosis and C. neoformans.

•Lymphocytes are also found in the C.S.f in trypanosomlasis meningoencephalitis and neurosyphilis.

•Eosinophilic, when the CSf contain mainly eosinophilus. This rare form of meningitis is caused by helminthes larvae such as Angiostrongylus cantonensis and Dirofilaria imitis

•Meningitis of the newborn (neonatal meningitis) is caused mainly by E.coli, S. agalactiae (Group B), S. avreus, L. Monocytogenes.

•Haemophilus meningitis occurs mainly in infants and young children.

Commensals

No normal microbial flora

Collection of Csf

•It should be collected by medical officer in aspectic procedure

•The fluid is usually collected from the arachnoid space. A sterile wide-bore needle is inserted between the 4th and 5th lumbar vertebrate and C.s.f. is allowed to drip into a dry sterile container.

159

•A delay in examining C.S.f reduces the chances of isolating a pathogen.

•It will also lead to a falsely low glucose value due to glycolysis. If typanosomes are present, they will not be found because they are rapidly lyzed once the C.S.f has been withdrawn.

1)Take two sterile, dry, screw-cap containers and label one No 1 (first sample collected, to be used for culture), and the other No 2 (second sample collected, to be used for other investigations).

2)Collect about 1ml of C.S.f in container No 1 and about 2-3ml in container No2

3)Deliver immediately the samples with a request form to the laboratory.

Note: It is necessary to culture the C.S.f in the health center because pathogens such as N.meningitidis, H. influenzae and S. pneumoniae, are unlikely to survive transport to the bacteriology laboratory.

•If unable to perform a cell count and estimate the protein and glucose, transfer C.S.f sample No 2 to a screw-cap

bottle containing sodium fluoride exalate and mix

This will preserve the cells and prevent the breakdown of glucose. Protein can also be estimated from a floride oxalated specimen.

Laboratory examination of C.S.f

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•It should be performed without delay and should be reported micro-organism especially Gram smear. The fluid should be handled with special care because it is collected by lumbar puncture and only a small amount can be withdrawn.

Two samples of C.S.f

•It is usual to use sample No 1 for the culture,

and sample No 2 for the cell caunt, microscopy and biochemistry.

This is because sample No 1 may contain blood (due to a traumatic lumbar puncture) which will affect the accuracy of the cell count and biochemical estimations.

I.Describe the appearance of the specimens Report:

•Whether the C.S.f is clear, slightly cloudy, cloudy or definitely purulent (looking like pus).

•If the C.S.f is purulent or markedly cloudy, make immediately a smear for gram staining, and report it as soon as possible.

•Whether it contain blood and if so whether sample No 2 contains as much blood as sample no 1.

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If blood is present in the C.S.f due to a traumatic lumbar puncture, sample No 1 will usually contain more blood than sample No 2.

If the blood is due to haemorrahage in the CNS, the two samples will probably appear equally blood-stained. Following a subarachnoid haemorrhoge the fluid may appear xanthochromic, ie. Yellow-red (after centrifuting) The fluid may also appear xanthromic if the patient is jaundiced or when there is spinal constriction.

-	Whether it contains clots

•Clotted C.S.f indicate an increase in fibrinogen.

•It occurs when there is spinal construction

•Clots can also be found in C.S.f. from patients with pyogenic meningitis (sterile beads to break the clot)

•In tuberculosis meningitis, if the C.S.f is allowed

to stand for several hours; skin (spider web clot) may form on the surface of the fluid.

This should be transferred to a slide, pressed out, alcohol- fixed, and stained by the Ziel-Neelsen method I.

2.Perform a cell count (1:2) value 5x106cell/l (sample No 2)

3.Test the specimen biochemically

-Glucose estimation ½ - 2/3 of that found in blood, i.e 2.5 – 4.0 mmol/l (45-72mg%)

-Low Glucos →pyogenic meningitidis

-Normal →viral miningitis.

162

-High glucose → hyperglycemia

-Total protein estimation and globulin test Value: 0.15 – 0.40g/l (15-40mg%)

•A positive pandy’s test for increase in total C.S.f protein in all form of meningitis.

•When the total protein exceeds 2.8/l

(200mg%), the fibrinogen level in usually increased sufficiently to cause the C.S.f to clot. (sever pyogenic meningitis)

4.Culture the specimen (sample No 1)

It is necessary, if the fluid contains cells and, or, the protein concentration is abnormal. Note: C.S.f should be cultured as soon as possible after collection.

If a delay is unavoidable, the fluid should be kept at 35-370C (never refrigerated).

If the C.S.f appears only slightly cloudy, centrifuge it in a sterile tube for 15-20minute and use the sediment for inoculating the plates.

Routine

163

Chocolate (heated blood) agar

Inoculate -N. meningitides -S. Preumonia -H. influential

Incubate in a CO2 enriched atmosphere at 35-370C for 48hours, cheeking for growth after overnight incubation.

Additional

MacConkey and blood agar if the patiente is a newborn infant incubate both plate at 35-370C overnight

-E.coli or other coliform

-S. agalacteae (Group B)

-Lesteriae monocytognes

-S. aureus

Lowenstein Jensen medium if tuberculous meningitis is suspected Sabourand agar if cryptococcal meningitis is suspected.

If capsulated yeast cells are seen in the microscopial preparations, inoculate a plate of sabouraud agar. Incubate at 35-370C for up to 72hours, cheeking for growth after overnight incubation.

5. Microscopy

The microscopical examination of C.S.f is required if the specimen appears abnormal, contains cells and, or, the total protein is raised with a positive pandy’s test.

Routine:

164

Gram smear

•Gram negative intracellular diplococci that could be N. meningitidis.

•Gram positive diplococci or short streptococci that could be S. pneumoniae. It is often possible to see the capsules as unstained are as around the bacteria.

•Garm negative rods, possibly H. influezae

•Gram negative rods, could also be E.coli or other coliforms, especially if the C.S.f is from a newborn infant.

•Gram positive cocci in groups and singly, possible S.aureus.

•Gram positive streptococci, possibly S. agalacteae (G -.B)

•Gram positive yeast cells, C. neoformans.

Additional

Ziel-Neelsen – smear

M. tuberculosis

Indian ink preparation if cryptococcal meningitis is suspected: Wet preparation to detect amoebae or Trypanosome Giemsa stain to detect morulla cells or Burkett’s lymphoma cells

These can be found when trypanosomes have invaded the CNS.

•They are larger than most lymphocytes, stain dark red mauve and contain vacuoles.

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•Morula cells contain Igm and are throught to be degenerate plasma cells.

Collection, transport and Examination of Blood AND Bone marrow

Possible pathogens

Gram positive	Gram negative
S. aureus	Salmonella typhi
Viridans streptococci	Other salmonella
S. Pneumoniae	Brucella species
S. pyogenes	H. influenzae
Enterococci	P. aeruginosa
Anaerobic streptococci	Klebsiella strains
Clostridium prefringes	E. coli
	Proteus species
	Bacteriode species
	Neisseria meningitidis
	Yersinia pestis

Fungi: Candida albicans and other yeast Cells and other systemic mycosis

Parasites: Plasmodium species (malaria parasites) Trypanosoma species Wuchererila bancrofti

Brugia species Loa loa Leishmania donovani

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Note:

1.The presence of bacteria in the blood is called bacteraemia. The term septicaemia refers to a severe and often fatal infection of the blood in which bacteria multiply and release toxins in to the blood stream.

2.Bacteria that are associated with neonatal septicaemia include E.coli and other coliforms, spaphylococci and B.haemolytic Group B streptococci.

3.In typhoid, salmonella typhi can be detected in the blood of 75-90% of patients during the first 10 days of infection and in about 30% of patients during the third week.

•In chronic salmonellosis, bacteria are often more rapidly and successfully isolated from bone marrow, especially once antimicrobial treatment has been started.

4.Y. pestis can be isolated from the blood in septicaemia plague. The organism in highly infectious.

Commensal

Neither blood nor bone has a normal microbial flora.

Collection and culture of Blood and Borne marrow

9Blood and bone marrow require culturing immediately

after collection, before clotting occurs. Choice of culture media

9Because septicaemia is such a serious condition, it is essential to use media that will provide the fastest

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growth and isolation of as wide a range of pathogens as possible.

9The following media are suitable for routine culture of blood and bone marrow:

-Tryptone soya (tryptic soy) diphasic medium

-Thioglycollate broth medium

Tryptone soya (tryptic soy) diphasic medium

¾A diphasic (two phase) medium is one that combines an agar slope with a broth medium.

Because the bacteria can be seen growing on the slope, the

need to subculture on a solid medium every few days is avoided, thus reducing the risk of contamination.

¾Tryptone soya diphasic medium consists of a tryptone soya agar slope and a tryptone soya broth to which is added Liquoid and P-aminobenzoic acid.

Liquoid: It is the commercial name for sodium polyanethol

sulphonate. It prevents clotting of the blood and neutralize the natural bactericidal substances in fresh blood.

P. Aminobenzoic acid: This neutralizes the action of sulphonamides should these be present in the blood.

¾Tryptone soya diphasic medium is suitable for the growth of a wide range of pathogens.

Incubation in CO2 is required for the culture of brucella species. Strict anaerobes will not grow in this medium.

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Thioglycollate broth

¾This consists of a nutrient broth to which is added thiogiycollate to provide the condition necessary for the growth of anaerobes. Most aerobic bacteria will also grow in thioglycollates broth.

¾Because liqoid is not added to this medium, a sufficient volume of broth must be used to prevent the blood from clotting and to dilute out the blood’s natural bactericidal substances.

The blood should be diluted at least 1 in 10 in the broth.

Examination of Blood and Bone marrow

1.Collect and culture the specimen

Blood

•It should be collected before antimicrobial treatement has been started and at the time the patient’s temperature is beginning to rise.

•To increase the chance of isolating a pathogen, it is usually recommended that at least two specimens (collected at different times) should be cultured.

•Blood for culture must be collected as aseptically as possible.

2.Insert the needle through the rubber line of the bottle cap and dispense 5ml of blood into each culture bottle.

3.Gently mix the blood with the bruch.

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•The blood must not be allowed to clot in the culture

media because any bacteria will become trapped in the Culture medium.

Incubate the inoculated media:

Thioglycollate broth

At 35-370C for up to 2 weeks, examining and sub-culturing

•Look for visible signs of bacterial growth such as turbidity above the red cell layer, colonies growing on top of the red cells (“cotton balls”), haemolysis, gas bubbles and clots.

•A sterile culture usually remains clear

•If there are signs of bacterial growth, subculture the broth and examine a gram stained smear for bacteria

Tryptone soya diphasic medium

At 35-370C for up to 4 weeks.

•Look for colonies on the agar slope (preferably using a hand lense), and signs of bacterial growth in the broth.

•Colonies of staphycococ, s .typhi, brucellosis and most coliforms can usually be seen easily. Where as colonies of pneumococci, S. pyogens, and Y.pestis are not easily seen.

•If growth is present subculture to blood agar plate, Chocolate agar plate, MacConkey

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Collection, transport and examination of effusions (synovial, pleural, pericardial, ascitie and hydroceles fluids)

¾An effusion is fluid which collects in a body

cavities

Fluid which collects due to an inflammatory process is referred to as an exudates and that which forms due to a non-inflammatory condition is referred to as a transudates.

¾If the effusion is all exudates, it is important to investigate whether the inflammatory process is all infective one.

¾Effusions sent to the laboratory for investigation include:

Fluid	Origin
Synovial	From joint
Pleural	From the pleural cavity (space between the
	lungs and the inner chest wall)
Pericardial	From the pericardial sac (membranous sac
	surrounding the health)
Ascitic (peritoneae)	From the peritoneal (abdominal) cavity
Hydrocele	Usually from the sacs surrounding the tests.

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1.Synovitis means inflammation of the synovial membrane (living of a joint capsule). It can be caused by bacteria,rheumatic disorder or injury.

2.Inflammation of a joint is called arthritis.

The term polyarthritis is used when many joints are affected. Arthritis may be caused by bacteria (infective arthritis), rheumatoid arthritis, gout and pseudogout, osteoatrhtitus

3.The term pleural effusion is used to describe a non-purulent serous effusion which sometimes forms in pneumonia, tuberculosis, malignante disease etc

Empyema is used to describe a purulent pleural effusion when pus is found in the pleural space.

4.Peritonitis means inflammation of the peritoneum, which is the serous membrane that lines the peritoneal cavity. Ascites refers to the accumulation of fluid in the pentional cary causing abdominal swelling.

Commensales

No microbial flora

Collection is carried out by a medical officer

-2-3ml without anticogulent, to see whether clotting occurs.

-9ml which contain 1ml sterile sodium citrate (3g/l (3% w/v) solution.

-do the csf:

-cell cout

-protein estimate

-microscopy

-culture

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CHAPTER THREE

Learning Objective

1.At the end of the lesson, the student shall be able to:

•List the antigenic structure of bacteria

•Apply the different chemical laboratory methods to identify the pathogenic bacteria

•Develop the major classification scheme for gram-positive and gram-negative bacteria

GRAM POSITIVE COCCI

Genus Staphylococci

Genus Streptococci

2.1.1. GENUS: STAPHYLOCOCCI

Characteristics:

•Gram positive non spore-forming non-motile, spherical cells, usually arranged in grape-like clusters

•Single cocci , pairs, tetrads and chains are seen in liquid cultures

•Young cocci stain strongly gram-positive, on aging many cells become gram-negative

•The three main species of clinical importance

. Staphylococcus aureus

. Staphylococcus epidermidis

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. Staphylococcus saprophyticus

Less common staphylococcal species

. Staphylococcus lugdenensis

. Staphylococcus hominis

. Staphylococcus warneri

•Can readily grow in ordinary media under aerobic and micro- aerophilic conditions

•grow most rapidly at 37 0c but form pigment best at room temperature of 20-25 oc

•Colonies in solid media are round, smooth, raised and glistening.

•Some of them are normal flora of the skin and mucus membrane of human, otrhhers cause suppuration abscess

formation and fatal septicemia

•Produce catalase, which differentiate them from the streptococci.

•relatively resistant to drying , heat, and 9% NacI, but readily inhibited by 3 % hexachlorophene

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Fig 3.1 Staphylococci

Antigenic structure:

1.Peptidoglycan( Mucopeptide): Polysaccharide polymer which provide the rigid exoskeleton of the cell wall. It is important in the pathogenesis of infection like eliciting production of cytokines and opsonic antibodies; chemoattractant for polymorphs;and activate complement

2.Teichoic acid: Polymer of glycerol or ribitol phosphate

3.Protein A: Important in immunologic diagnostic test (coagglutination test).

4.Capsule: Anti-phagositic property

5.Enzymes

. Catalase- Produced by staphylococci Converts H202 into H20 and 02

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Catalase test differentiates staphylococci(catalase-positive) from streptococci(catalase-negative)

. Coagulase and clumping factor

. Coagulase clots oxidated or citrated plasma. Coagulase may deposit fibrin on the surface of organism and alter ingestion by phagocytic cells.

. Clumping factor: A surface compound that is responsible for adherence of the organism to fibrinogen and fibrin

Produced by Staphylococcus aureus

Determines Invasive potential of the organism. Coagulase test differentiates S.aureus(coagulase-

positive) from S.epidermidis (coagulase-negative)

. Hyaluronidase-Spreading factor

. Proteinases and lipases

. Staphylokinase- Fibrinolysin

. β-lactamase-Provides resistance of staphylococcus to β-lactam antibiotic like penicillin.

. Dnase: Deoxyribonucleotidase

. Nuclease

6.Toxins

. Exotoxins(α, β, γ, δ)

. Enterotoxin-Produced by S.aureus when grown in carbohydrate and protein foods.

Multiple (A-E, G-I, K-M) soluble heat-stable, gut enzyme resistant toxins which act on neural receptors

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in the gut to stimulate vomiting center in the central nervous system. It is superantigen causing staphylococcal food poisoning

. Toxic shock syndrome toxin- Superantigen desquamative toxin Produced by S.aureus and Causes fever, shock, multiple-organ failure and skin rash.

. Exfoliative toxin-Epidermolytic superantigen produced by S.aureus and uses generalized desquamation of the skin (staphylococcal scalded skin syndrome).

Epidermolytic toxin A: Chromosomal gene product and heat stable Epidermolytic toxin B: Plamid mediated and heat labile

. Leukocidin: S aureus toxin which kills WBCs by forming pores and incresing cation permeability

Clinical features:

. Folliculitis:Infection of one hair follicle.

. Curbuncle: Infection of multiple hair follicle and surrounding skin.

. Cellulitis: Infection of skin and subcutankeous tissue.

. Abscess formation: focal suppuration

. Mastitis: Infection of breast, especially in lactating mother

. Bulous impetigo: Crusted superficial skin lesion

. Pneumonia: Infection of lung parenchyma.

. Empyema: Accumulation of pus in pleural space

. Osteomyelitis: Infection of bone

.Endocarditis and meningitis: Infection of heart tissue and leptomeninges respectively.

. Food poisoning: Caused by enterotoxin produced by S.aureus

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. Characterized by violent nausea, vomiting, and diarrhea

. Toxic shock syndrome: Caused by toxic shock syndrome toxin-1 produced by S.aureus

. Characterized by abrupt onset of high fever, vomiting, diarrhea, myalgia, scarlatiform rash,and hypotension with cardiac and

renal failure in the most severe disease

. Occurs with in 5 days after the onset of menses in young women who use tampoons

. Staphylococcal scalded skin syndrome: Caused by exfoliative toxin produced by S.aureus.

S. saprophyticus: Relatively common cause of urinary tract infections in young women

S. epidermidis: occasional cause of infection often associated with implanted appliances and devices

Laboratory Diagnosis:

Specimen: Surface swabs, pus, blood, sputum, cerebrospinal fluid

Smear: Gram positive cocci in clusters, singly or in pairs.

Culture: Grow well aerobically and in a CO2 enriched ordinary media at an optimal temperature of 350c-370c.

Colony appearance:

S.aureus: characteristically golden colonies. frequently non-pigmented after over-night incubation. hemolytic on blood agar plate.

7.5% Nacl containing media is used for mixed flora contaminated specimen

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Mannnitol slt agar is used to screen for nasal carriers of S. aureus S.epidermidis: white colonies, non-hemolytic S.saprophyticus: may be white or yellow, non-hemolytic.

Biochemical reaction 1. Catalase test

Active bubbling…………….Catalaseproducing Bacteria (Staphlococci)

No active bubbling…………Non-catalase producing bacteria (streptococci)

2. Coagulase test

a. Slide test: To detect bound coagulase Clumping with in 10 seconds………… S.aureus

No clumping with in 10 seconds………CONS(Coagulase negative

staphylococci)

c.Tube test: To detect free coagulase Fibrin clot…………….S.aureus

No fibrin clot…………CONS Sensitivity testing:

Novobiocin sensitive……. S.aureus and S.epidermidis Novobiocin resistant……...S.saprophyticus

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Table 2.1 DIFFERENTIATION OF SPECIES

Organism Colony	Catalase	Coagulase	Novobiocin Hemolysison
appearance	Production	Production	sensitivity	Blood agar
S. aureus	Golden	Positive Positive	Sensitive	Positive
S. epidermidis	White-Gray	Positive Negative	Sensitive	Negative
S. saprophyticus	White-Gray	Positive Negative	Resistant	Negative

Treatment

Penicillin sensitive staphylococci………penicillin, ampicillin

Penicillin resistant staphylococci………cloxacillin, Nafcillin

Methicillin resistant staphylocicci……… Vancomycin

Prevention and control

Source of infection is shedding human lesions, the human respiratory tract and skin

Contact spread of infection occur in hospitals

Treatment of nasal carriers with topical antiseptics or rifampin and anti-staphylococcal drug

2.1.2.GENUS: STREPTOCOCCI Characteristics:

•They are non-motile, non-sporulating, gram- positive facultative anaerobes

•Spherical or oval cells characteristically forming pairs or chains during growth

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•Grow well on ordinary solid media enriched with blood, serum or glucose.

•Most streptococci grow in solid media as discoid colonies

•Capsular streptococcal strains give rise to mucoid colonies

•They are aerobic bacteria in which growth is enhanced with 10% carbondioxide.

•They are catalase-negative.

•They are widely distributed in nature and are found in upper respiratory tract, gastrointestinal tract and genitourinary tract as normal microbial flora.

•They are heterogeneous group of bacteria, and no one system suffices to classify them.

•The currently used classification is based on colony growth characteristics, pattern on blood agar, antigenic composition of group specific cell wall substance and biochemical reaction

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