INTRODUCTION

Microbiology
Is a subject which deals with living organisms that are individually
too small to be seen with the naked eye These organisms include bacteria, algae, protozoa, fungi, and virus

Sub divisions of microbiology

Bacteriology deals with bacteria Mycology- deals with fungi Phycology - deals with Algae Protozology deals with Protozoa. Virology and Rickettisiology -studies about viruses and Rickettisia

Medical Bacteriology

The study of bacterial pathogens, the disease caused by them, and the bodys defenses against these diseases.

Microbes natural distribution

Microorganisms can be found nearly everywhere as normal inhabitants of the earth (biosphere) They exist in soil, water, air, in our food, in our clothing, in our body etc Can also survive in most unlikely environment like in cold air, in hot springs at temperatures of 90 oC Inhabit the surface of living human and animal bodies and grow abundantly in the mouth and intestinal tract A small percentage microbes are pathogenic The others are considered beneficial or harmless, or Opportunistic - cause disease only if they accidentally invade the wrong place at the right time such as when the host immunity is low Normal flora - live on the human body without causing disease and apparent physiological response
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Basic structure of cells Prokaryotic

More primitive, small and without organelles, nuclear materials (DNA) NOT enclosed by membrane Genetic material not organized into chromosome Divide by binary fission Ex. Bacteria, Rickettsia, Mycoplasm More advanced, larger & contain organelles Ex: Animals, plants, fungi, protoza Nucleus within a nuclear membrane Have organelles with numerous discrete membranes Divide by mitosis

Eukaryotic

Basic features of Bacterial Cell

Typical prokaryotic cell Contain both DNA and RNA Most grow in artificial media Replication is by binary fission Contain rigid cell wall

Bacterial cell wall

a non-living secretion of the cell membrane, Multi layered structure and constitutes about 20% of the bacterial dry weight Average thickness is 0.15-0.5 m

Chemical Composition of cell wall

The major component of cell wall is peptidoglycan (PG) The rigidity of the cell wall is due to the presence of this a unique substance called peptidoglycan layer (murein) It is composed of N-acetyl Muramic acid and N-acetyl Glucosamine backbones cross-linked with peptide chain and pentaglycine bridge This PG is found only in bacteria and not found in other Micro organisms Some times called back bone of bacteria.

The gram stain divide bacteria in to two classes, which differ in their ability to retain a basic dye after fixation.

The difference is based on major variation in the structure of the cell wall.

Types of Cell Wall

I.

Gram positive cell wall of bacteria has two layers (Peptidoglycan (PG) cross linked with teichoic acid) The PG layers is much thicker than Gram negative bacteria and is 15 50 nm thick

II. Gram negative cell wall of bacteria Is some what complex than Gram positive bacterial cell wall Has thin peptidoglycan layer (3 8nm) Has high lipid content (lipopolysaccharied) in the outer membrane Has periplasmic space.

CLASSIFICATION OF BACTERIA

1. 2. 3. 4. 5. 6. 7. 8. 9.

Bacteria are classified in to 19 different categories in Bergeys manual of determinative bacteriology, 8th (1974), and the classification is based on Morphology Staining Motility Growth Nutritional requirement Bio chemical and metabolic activity Pathogenecity Amino acid sequencing of proteins Genetic composition

Morphology: - Bacteria vary widely in size, ranging from 0.2 um to 10um long -There are there basic shapes 1. Spherical or coccoid/cocci- (singular coccus) 2. Rods or bacilli (singular - bacillus) 3. Spirals or spirilla (Singular - Spirillum)

Fig . Different bacterial morphologies A. Cocci, B. Bacilli, C. Spiral shape

The cells of cocci may be found in various arrangements depending on the species and the way they divide e.g Micrococcus:-Cocci occurring singly. - Diplococci- Pairs of cocci - Strepto cocci Cocci in chain - Staphylococci- Cocci in cluster - Tetrads Four cocci as in box - Octads(sarcina) Eight cocci as in box

Bacilli (rods) may be short or long, thick or thin, pointed or with blunt ends, - Some rods resemble cocci and are often called coccobalilli because they are very short small bacilli. Some bacilli stack up next to each other eg. Diphteroids, Some are coma shaped e.g. V.cholara Spiralls usually occur singly. -The different species of spirilla varies in size, length, rigidity, number and amplitude of their coils.

Fig. different bacterial arrangements

Making smear is a precondition to facilitate staining and further observation of microorganisms under microscope. Without making appropriate smear, which is thin enough to make a single layer of bacteria, it is difficult to observe and read different staining reactions of bacteria. Even with the microscope, bacteria are difficult to see unless they are treated in a way that increases contrast between the organisms and their background. The most common method to increase contrast is to stain part or all of the microbe.

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Differential staining method A method in which multiple stains (dye) are used to distinguish different group of bacteria. e.g. Grams stain, Ziehl-Neelson stain.

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GRAMS STAIN This method was developed by the Danish bacteriologist Christian Gram in 1984.
Basic concepts: Most bacteria are differentiated by their gram reaction due to differences in their cell wall structure. The surface of bacterial cell has got a negative charge due to the presence of polysaccharides and lipids (PG) this has made the surface of the bacteria to have affinity to cationic or basic dyes (when the colouring part is contained in the basic part.)

Principle The principle of Grams stain is that cells are first fixed to slide by heat or alcohol and stained with a basic dye (e.g. crystal violate), which is taken up in similar amounts by all bacteria. The slides are then treated with a Grams iodine to fix (mordant) the crystal violet stain on Gram positive bacteria, decolorized with acetone or alcohol, and finally counter stained with Safranin.

Gram positive bacteria: - stain dark purple with crystal violet and are not decolorized by acetone or ethanol. The following are some important examples of gram positive bacteria.

Staphylococcus, Streptococcus, Clostridium Bacillus Corynebacterium etc

N.B.

The reason for the retention of the primary stain (CV) by the gram positive bacteria after decolorization is due to the presence of more acidic protoplasm (PG layer) of these organisms which bind to the basic dye.

Gram negative bacteria: - stain red because after being stained with crystal violet they are decolorized by acetone or ethanol and take up red counter stain. (Neutral red, Safranin or dilute carbol fuchsin). The following are some important gram negative bacteria:

Nesseria spp. Haemophilus spp. Salmonella, shigella, vibrio, Klebsilla, Coliforms etc.

Required reagents Crystal violet Grams Iodine Acetone-Alcohol or 95% Alcohol Safranin or Neutral red

Figure: Gram positive and Gram negative bacteria

Ziehl-Neelson (Acid fast Bacilli-AFB) staining method

Developed by Paul Ehrlich in1882, and modified by Ziehl and Neelson Ziehl-Neelson stain is used for staining mycobacteria which are hardly stained by Grams staining method. Once the Mycobacteria is stained with primary stain it can not be decolorized with acid, so named as acid-fast bacteria.

- Mycobacteria typically are slightly bent or curved slender rods. - The most striking chemical feature of mycobacteria is their extra ordinary high lipid content in the cell wall (up to 60% of its dry weight). This high lipid content probably accounts for some of the other unusual properties of mycobacteria.
E.g Relative impermeability to stains, acid fastness, unusual resistance to killing by acid and alkali.

The cell wall of Mycobacteria also contains a peptidoglycan layer, glycolipids, protein and Mycolic acid(This is unique to mycobacteria, nocardiae and corynebacteria).

Principle of Ziehl-Neelson (Acid fast) staining method Sputum smear is heat fixed, flooded with a solution of carbilfusin (a mixture of basic fuschin and phenol) and heated until steam rises. The heating which facilitate penetration (entrance) of the primary stain into the bacterium. After washing with water, the slide is covered with 3% HCl (decolourizer). Then washed with water and flooded with methylene blue ( Mycobacterium tuberculosis) and malachite green (Mycobacterium leprae).

Ziehel Neelson (AFB) stains

Carbolfuchsin 3% Acid alcohol Methylene blue (malachite green)

Hot and cold Ziehel Nelson technique In the hot Zn technique the phenolic carbol fuchsin stain is heated to enable the dye to penetrate the waxy mycobacterial cell wall. Techniques that do no heat the stain are referred to as cold techniques. In these, a penetration of the stain is usually achieved by increasing the concentration of basicfuchsin and phenol. Comparison between the hot and cold method has shown that both M. leprae and M. tuberculosis stain less well by the cold method.

Fluorescence staining for Mycobacteria

Fluorescence microscopy uses illumination from either a quarth halogen lamp or a high pressure mercury vapor lamp. The advantage of fluorescence microscopy is that a low magnification is used to scan smears, allowing a much larger of the smear to be seen and resulting in more rapid examination.

Auramine O 3% acid alcohol Potassium permanganate

Bacterial Growth

Each bacterial species has a specific tolerance range for specific environmental parameters rate of bacterial growth are greatly influenced by the following environmental parameters 1- Nutrition 2- Temperature 3- Oxygen 4- PH 5- Salentery 6- Pressure 7- Light radiation

Oxygen requirement

The need of oxygen for particular bacterium reflects its mechanism to meet the requirement of energy. On the basis of this requirement, bacteria have been divided in to:

Obligate Anaerobes-these grow only in the environment devoid of oxygen e.g. clostridium Facultative aerobes- these can grow under both aerobic and anaerobic conditions, e.g. enterobacteriaceae Obligate aerobes- these cannot grow unless oxygen is present in the medium e.g. pseudomonas Microaerophilic- these organisms can grow under conditions with low oxygen tension e.g. clostridium titani. Aerotolerant anaerobes These bacteria oxidize nutrient substrates without using elemental oxygen. Although, unlike obligate anaerobes, they can tolerate it.

Respiration (Aerobic)

a type of heterotrophic metabolism that uses oxygen and in which 38 moles of ATP are derived from the oxidation of 1 mole of glucose In aerobic respiration, molecular 02 serves as the terminal acceptor of electrons. Most aerobic organisms oxidize glucose another type of heterotrophic metabolism, an organic compound rather than oxygen is the terminal electron (or hydrogen) acceptor. NO3-, SO4 2-, CO2, or fumarate can serve as terminal electron acceptors (rather than 02), depending on the bacterium Less energy is generated from this incomplete form of glucose oxidation, but the process supports anaerobic growth.

Fermentation (Anaerobic)

Bacterial growth curve

The growth cycle of bacteria has four major phases. I. The Lag phase II. The log phase (exponential phase) III. The stationary phase IV. The decline phase If a small number of bacteria are inoculated into a nutrient medium and the bacteria are counted at frequent interval, the typical phase of a standard growth curve can be demonstrated.

Host Parasite relationship

I.
II.

III.
IV.

Symbiosis: The ability to live in the tissues of the host with mutual benefit. Neither of them are harmed. Commensalism: The ability to live on the external or internal surface of the body with out causing disease. Eating at the same table Parasitism: one ( the host) is harmed and the other(the parasite ) is benefited Pathogenicity: The ability of an organism to cause disease.
The outcome of the host- parasite relationship depends on a balance between
-

the virulence of the parasite and the resistance of the host.

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General Concepts Host Susceptibility Resistance to bacterial infections is enhanced by phagocytic cells and an intact immune system. Initial resistance is due to nonspecific mechanisms. Specific immunity develops over time. Susceptibility to some infections is higher in the very young and the very old and in immunosuppressed patients.

Bacterial Infectivity Bacterial infectivity results from a disturbance in the balance between bacterial virulence and host resistance. The "objective" of bacteria is to multiply rather than to cause disease; it is in the best interest of the bacteria not to kill the host.

Host Resistance Numerous physical and chemical attributes of the host protect against bacterial infection. These defenses include the antibacterial factors in secretions covering mucosal surfaces and rapid rate of replacement of skin and mucosal epithelial cells.

Bacteria invading tissues encounter phagocytic cells that recognize them as foreign, and through a complex signaling mechanism involving interleukins, and complement, mediate an inflammatory response in which many lymphoid cells participate.

Genetic and Molecular Basis for Virulence

Bacterial virulence factors may be encoded on chromosomal, plasmid, transposon, or temperate bacteriophage DNA; virulence factor genes on transposons or temperate bacteriophage DNA may integrate into the bacterial chromosome.

Table: Genetic basis for virulence selected bacterial pathogens

Culture Media
Are artificially prepared media containing the required nutrients used for propagation of micro organisms. Once the bacteria are grown we can: 1. Identify them either by presumptive lab diagnosis like Gram stain or by definitive lab diagnosis like biochemical test 2. Test the antimicrobial sensitivity of the bacteria (drug testing). This helps to know whether the bacteria are sensitive or resistant to known antimicrobial drugs.

Types of culture media The main types of culture media are: 1. Basic media 2. Enriched and enrichment media 3. Selective media 4. Indicator (differential) media 5. Transport media

1. Basic Media These are simple media that will support the growth of micro organisms that do not have special nutritional requirements. Example - Nutrient Agar - Nutrient broth Purposes of basic media 1. Are used in preparation of enriched media. 2. Are used to maintain stock culture. 3. For sub culturing pathogens from differential and selective media prior to performing biochemical and serological identification tests.

2. Enriched/Enrichment media

These media are required for growth of organism with extra nutritional requirements such as H. influenza, Neisseria spp, and some streptococcus species. An enriched medium increases the number of a pathogen by containing all the necessary ingredients to promote its growth. The media can be enriched with whole blood, lyzed blood, serum, vitamins, and other growth factors. E.g: - Blood Agar (contain whole blood) - Chocolate agar (contain lyzed blood) Enrichment media: this term is usually applied to fluid selective media E.g. Tryptosoya broth Selenite F broth

3. Selective media

These are solid media which contain substances which inhibit the growth of one organism to allow the growth of the other to be more clearly demonstrated. The medium is made selective by incorporation of certain inhibitory substances like bile salt, crystal violet, antibiotics, etc.

E.g. Addition of crystal violet favors the growth of gram negative bacteria and slows the growth of Gram positives.

Selective medium is used when culturing a specimen form a site having normal microbial flora to prevent unwanted contaminants overgrowing a pathogen. E.g.1. Thiosulphate citrate bile salt sucrose agar(TCBS) - is alkaline medium and selective for V. cholera.

2. Xylose Lysine Deoxy Cholate (XLD) agar - selective for Salmonella and Shigella. 3. Modified New York City (MNYC) medium - Selective for Neisseria gonorrhoeae. 4. Butzler medium - Selective for Campylobacter species. 5. Salmonella and Shigella agar (SSA) - Selective for Salmonella and Shigella species.

5. Differential (Indicator) media These are media to which dyes or other substances are added to differentiate micro-organisms. Many differential media distinguish between bacteria by incorporating an indicator which changes colour when acid is produced following fermentation of a specific carbohydrate. E.g. Mac Ckonkey agar - contain neutral red as an indicator and lactose as carbohydrate. Lactose fermenting bacteria will become pink and other bacteria become colourless.

Figure: differential media (MacConkey agar media , pink colonies are lactose fermenters)

6. Transport media These are mostly semisolid media that contain ingredients to prevent the overgrowth of commensals and ensure the survival of aerobic and anaerobic pathogens when specimens cannot be cultured immediately after collection. Their use is particularly important when transporting microbiological specimens form health centres to the district microbiological laboratory or regional public health laboratory. Example 1.Cary-Blair medium : is used for preserving and transporting enteric pathogens. 2.Amies transport medium:- is used for transportation of gonococci.

Culture media can be classified by their consistency (form) as: Solid media Semi-solid media and Fluid media
A. Solid media Are solidified by agar Are used mainly in Petri dishes as plate cultures, in bottles or tubes as stab (deeps) or slope cultures. The purpose of culturing on solid medium is principally to isolate discrete colonies of each organism present in the specimen. this will enable pure cultures to be produced for identification and sensitivity testing.
NB: The colonial appearances and changes in solid media made by colonies may provide valuable identification information

B. Semi solid media

Are culture media prepared by adding small amount of agar (0.4 - 0.5% W/V) to a fluid medium. Semi solid media are used mainly as transport media, and for motility and biochemical tests.

C. Fluid culture media Fluid media are most commonly used as enrichment when organisms are likely to be few E.g. blood culture Is also used for biochemical testing E.g. Peptone water

Inoculating Techniques NB: For sputum and stool samples since they have large number of bacteria, prior to inoculation we have to dilute or homogenize the samples. 1. Using a sterile loop or swab of the specimen, apply the inoculation to a small area of the plate. 2. Flame sterilize the loop, when cool or using a second sterile loop, spread the inoculation systematically. This will ensure single colony growth

Figure. Inoculation techniques.

Inoculation of Slopes

To inoculate slopes use a sterile straight wire to streak the inoculation down the center of the slope and then spread the inoculation in a zigzag pattern as shown in the figure.

Figure. Inoculation of Slopes.

Labeling of inoculated media

Using grease pencil or marker, label inoculated media with the date and the patients number always label the base of the culture plate not lids.

Incubation of inoculated media Inoculated media should be incubated as soon as possible. A delay in incubation can affect the viability of pathogens. Micro organisms require incubation at the temperature, humidity & gaseous atmosphere most suited for their metabolism.

Temperature of Incubation

The temperature at which micro-organisms grow best is referred to as its optimum temperature. The temperature selected for routine culturing is 35 -37oC, however, most microbiologists recommend 35oC. Temperature of growth is also used in the differentiation of mycobacterium species.
E.g. No growth is produced by M. tuberculosis at 25oC where as many opportunistic and saprophytic mycobacteria grow at this lower temperature. Some exception is that Yersinia enterocolitica grows best at 20 -28oC which helps to identify the species. Humidity - growth atmosphere which is too dry can affect the growth and viability of many pathogens.

Appearance of Bacterial colonies on solid Media

Bacterial colonies should be examined in a good light and a low power magnifying lens can help to see morphological details. 1. When viewed form above: colonies may appear round, irregular crenated, or branching. They may be transparent or opaque and their surface may be smooth or rough, dull or shiny. Eg. The colonies of pneumococci have a ringed appearance.

2. When viewed form the side: Colonies may appear flat or raised in varying degrees some times with beveled edges or with a central elevation or depression.

3. When touched with wire loop: Some colonies are soft and easily emulsified such as S.aureus. Where as others are difficult to break up such as S. pyogens. 4. The colour of colonies: this also helps to identify bacteria, especially when using differential media containing indicators.
E.g. - V. cholera in TCBS agar appear Yellow - Corny bacterium diphteriae in Tellurite agar appear black.

Figure:TCBS Agar - Vibrio chol.

Changes which may occur in the medium when bacteria are cultured on solid agar.

These include hemolytic reactions, pigment production, color changes surrounding carbohydrate fermenting colonies, and blackening due to hydrogen sulphide production. * An example of pigment forming organism is pseudomonas aeroginosa which produces a yellow green color in media such as blood agar and MacConkey agar. * An examples of organism that produces a color change is Vibrio chorera which is sucrose fermenting, giving a yellow color in TCBS agar.

Blacking due to hydrogen sulphide production is seen with many Salmonella cultured in kligler iron agar(KIA). Hemolytic reaction in blood agar is seen with beta hemolytic Streptococci (complete hemolysis) and alpha hemolytic pneumococci(partial hemolysis)

Figure: Beta hemolysis

Figure: Alpha hemolysis

NB. Morphological appearance of colonies on blood agar can vary depending on the species of blood used. E.g. Horse, sheep, or goat blood.

Reporting culture results The manner of reporting culture depends on whether the specimen is either from a sterile site or from site with a normal microbial flora. Sites normally Sterile: Identify and report all bacteria isolated up to their genera. And if helpful identify the actual species using bio chemical tests. Sterile sites include blood, bone marrow, CSF, pleural and peritoneal fluids. NB. For isolating organism from sterile sites use culture media which is non selective, and enriched.

Sites Having a Microbial Flora: Interpretation requires patients clinical data to judge whether an isolate is a pathogen which causes the patients illness. The laboratory report should indicate those organisms for which isolation technique has been performed. For example when the pathogen have been isolated from feacal specimen cultured on selective medium like SSA (salmonella shigela agar). The report should state as no salmonella or shigella organism isolated Sites with normal flora include faeces (stool), sputum, skin, throat and nose swabs, vagainal, cervical, and urethral swabs. NB: For isolating organism from sites with normal flora selective media are much better than general media.

ANTIMICROBIAL SENSITIVITY (SUSCEPTIBILITY) TESTING

The test is used to measure the ability of the drug to inhibit or kill pathogens in vitro. I.e. it is used to select effective antimicrobial drugs. Sensitivity test is performed:

For organisms with variable antibiotic sensitivity (un predictable sensitivity) E.g. Shigella For non responding patients after taking adequate therapy. For patients whose immune system is depressed For relapsing cases (reappearance of disease)

Sensitivity test is not performed

If the bacteria is a normal flora contaminant If the culture is mixed. Sensitivity is performed on pure culture and For organisms with predictable sensitivity E.g. - S. Pyogens & N. meningitis are sensitive to penicillin - Proteus species are generally resistant to tetracyclines, so no need of sensitivity testing.

Sensitivity Testing Techniques Laboratory antimicrobial sensitivity testing can be performed using: 1. A dilution technique and 2. A disk diffusion technique

1. Dilution technique for sensitivity test - This technique can be done either on Agar or broth media. Principle: Graded amounts of antimicrobial 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 smallest amount of antimicrobial agent required to inhibit the growth of the test bacteria (MIC-minimum inhibitory concentration) or to kill the test bacterial (MBC- minimum bactericidal concentration).

Procedure 1. Prepare liquid or solid media 2. Add graded amount of antimicrobial agent (drug) 3. Inoculate with the pure culture 4. Incubate at specified temperature. 5. Read and take the last tube with no growth as MIC. - This MIC or MBC value is then compared with known concentration of drug obtainable in the serum or other body fluids, and then the likely clinical response can be assessed.

FIGURE: Broth dilution susceptibility test.

FIGURE: Broth dilution susceptibility test. The stippled tubes represent turbidity produced by bacterial growth. The MIC is 2.0 g/mL.

2. Disk Diffusion Sensitivity Testing - Disk diffusion techniques are used by most laboratories to test routinely for antimicrobial sensitivity. (Because it is simple, economical & reproducible)

Principle: A disk of blotting paper is impregnated with a known volume and appropriate concentration of an antimicrobial agent, and this is placed on a plate of sensitivity testing agar uniformly inoculated with the test organism. The antimicrobial diffuses from the disc into the medium and the growth of the test organism is inhibited at a distance from a disc that is related to the sensitivity of the organism. Strains sensitive to the antimicrobial agent are inhibited at a distance from the disc, where as resistant strains have smaller zones of inhibition or grow up to edge of the disc.

-The zone of inhibition is measured by clippers or ruler

and values are matched (compared) with the predetermined standard values and reported as susceptible, Intermediate and resistant.

Figure: disc diffusion technique

NB: WHO recommends the (NCCLS-modified Kirby Bauer disk diffusion technique.(NCCLS National committee for clinical laboratory standards)
Kirby-Bauer Modified Disk Diffusion The validity of this technique depends on use of reliable Muller Hinton agar, discs of correct antimicrobial content and turbidity standard equivalent to McFarlands.

Mueller Hinton Sensitivity testing Agar Prepare the medium as instructed by the manufacturer. The PH of the medium should be 7.2 7.4. Pour into 90mm diameter sterile Petri dishes to a depth of 4mm. (i.e. about 25ml of Mueller Hinton agar per plate] Care must be taken to pour the plates on a level surface so that the depth of the medium is uniform.

Control each new batch of agar by testing it with a control strain. E.g. E. faccalis (ATCC 29122 or 33186) and co trimoxazole disc. The zone of inhibition should be 20mm or more in diameter. The plates should be stored at 2 8oC for up to 2 weeks

Antimicrobial Discs The choice of antimicrobials to be included in sensitivity tests will depend on the pathogen, the range of locally available antimicrobials and local prescribing polices(National drug list). Most paper discs can be used for 1 year or longer from the date of manufacture if stored properly (2 8oC for working stock). About 1 hour before use, the working stock of discs should be allowed to warm to room temperature.

Turbidity standard equivalent to McFarland 0.5 This is a barium sulphate standard against which the turbidity of the test and control inocula can be compared.

Figure: equipments for susceptibility testing

Procedure: 1. Using a sterile wire loop, touch 3 5 well isolated colonies of similar appearance to the test organism and emulsify in 3 4 ml to sterile physiological saline or nutrient broth. 2. In a good light match the turbidity of the suspension to the turbidity standard (mix the standard before use) 3. Using a sterile swab, inoculate a plate of Muller Hinton agar. Remove excess fluid by pressing and rotating the swab against the side of the tube above the level of the suspension. - Streak the swab evenly over the surface of the medium in three directions rotating the plate approximately 60o to ensure even distribution.

4. With the Petri dish lid in place, allow 3 5 minutes for the surface of the agar to dry. 5. Using sterile forceps or multi disc dispenser, place the appropriate antimicrobial discs evenly distributed on the inoculated plate. - The discs should be about 15mm from the edge of the plate and no closer than 25mm from disc to disc. No more than 6 discs should be applied in 90mm dish. - Each disc should be lightly pressed down to ensure its contact with the agar. It should not be moved once in place.

6. Within 30 minutes of applying the discs, invert the plate and incubate it aerobically at 35oC for 16 18 hours. 7. After over night incubation, examine the control and the test plates. Using a ruler measure the diameter of each zone of inhibition in mm on the underside of the plate. the end point of inhibition is where growth starts.

Figure: Removing colonies from a primary culture plate to make a suspension of the test organism.

Figure: Checking the turbidity of the test suspension against the turbidity of a chemical standard.

Figure: Avoiding using too much inoculum by pressing and rotating the swab against the side of the tube

Figure: Swabbing the surface of the susceptibility testing agar. The plate is swabbed in three directions, rotating the plate approximately 60 to ensure even distribution.

Figure: Placing antimicrobial discs on the inoculated plate.

Figure: Measuring the zones of inhibition in mm. The end of inhibition is where growth starts.

Interpretation of Zone Size Using the interpretative chart, interpret the zones sizes of each antimicrobial and report the organisms as Resistant, Intermediate (moderately sensitive) or Sensitive (susceptible).

Resistant: A pathogen reported as resistant implies that the infection it has caused will not respond to treatment with the drug to which it is resistant irrespective of dose or site of infection. Intermediate: A pathogen reported as intermediately sensitive suggests that the infection it has caused is likely to respond to treatment when the drug is used in larger doses than normal or when the drug is concentrated at the site of infection.

Consideration should be given to using other drugs that may provide more optimal therapy.

Sensitive (Susceptible):- A pathogen reported as sensitive suggests that the infection it has caused is likely to respond to treatment when the drug is used in normal recommended doses.
NB: It is necessary to report the first and second choice of antibiotics for a patients infection unless the strain is resistant.