Laboratory Methods For Identification Of Bacteria

Bacteria are either identified in:1. A pathological specimen obtained from the patient (e.g. pus, sputum, urine,

blood, stools, etc.) depending on the site of infection.2. After been grown on artificial nutrient media.

Bacteria are then identified by:(I) Microscopic Examination:a. Examination of fresh samples used for demonstration of bacterial motility;

using hanging drop method.

b. Morphology and staining reactions of bacteria.

Commonly used stains are:1. Simple stains: e.g. methylene blue stain.2. Differential stains: e.g.i. Gram stain: 1) Primary stain: Methyl violet- Iodine mixture.2) Decolourization with Alcohol.3) Counter stain: Diluted carbol fuchin.

Results: Gram (+) Purple Gram (-) Red Difference - due to structure of cell wall

• Gram (+) Thick cell wall• Gram (-) Thin cell wall

a

ii. Acid fast stain: (Ziehl-Neelsen stain)Differential Stain - divides bacteria into 2 groups Acid - Fast Non Acid - FastUsed to identify organisms in the Genera Mycobacterium (high lipid and wax content in cell wall)1. Carbol fuchsin (Red)2. Decolourization by sulphuric acid 20%.3. Counter stain with Methylene blue.

Acid - Fast organism: Red as Mycobacterium tuberculosisNon Acid – Fast organism: Blue

iii. Special stains: e.g. Capsule stain, flagella stain, …..

(II) Cultural Characters:Bacteria need nutritive culture media to multiply in vitro.An undefined medium (also known as a basal or complex medium). It is a medium that contains:1- A carbon source such as glucose for bacterial growth 2- Water 3- Various salts needed for bacterial growth. Defined media (also known as chemically defined media or synthetic media). Classification of Media:Media can be classified into:1-Minimal media (simple medium):It contains the basic nutritive requirements, e.g. nutrient broths and agar media.

2- Selective media:

Selective media are used for the growth of only selective microbes.It contains antibiotics, dye, or specific chemicals that inhibits the growth of most types of microbe and stimulate the isolation of one type. Mannitol salt agar (MSA) which is selective for Gram +_ve bacteria. Blood-free, charcoal-based selective medium agar (CSM) for isolation of Campylobacter. Lowenstein- Jensen medium: enriched selective media for T.B. T.C.B.S (Thiosulphate- Citrate- Bile- Sucrose agar): selctive for Vibrio cholera due to alk. pH.

3-Differential media: Differential media or indicator media distinguish one microorganism type from another growing on the same media.Indicators (such as neutral red, phenol red, eosin y, or methylene blue) Examples of differential media include:Eosin methylene blue (EMB), which is differential for lactose and sucrose fermentation.MacConkey (MCK), which is differential for lactose fermentation.

4- Enriched media: Enriched media contain the nutrients required to support the growth of a wide variety of organisms, including some of the more fastidious ones.Blood agar : Is an enriched medium in which nutritionally rich whole blood supplements the basic nutrients. It contains 5-10% human or animal blood.It shows the type of haemolytic activity of bacteria (complete, partial or non- haemolytic). Complete PartialHaemolysis of HaemolysisRBCs of RBCs(Beta (Alpha Haemolytic HaemolyticStreptococci) Streptococci)

Chocolate agar (heated blood agar):is enriched with heat-treated blood (40-45°C).

Lofflers serum media:Horse serum + glucose in a ratio 3:1It is used for cultivation of Corynbacterium diphtheria.

5- Transport media:

Transport medium is a simple organic medium that Maintain the viability of all organisms in the specimen without altering their concentration.

This type of medium mainly used for Temporary storage of specimens being transported to the laboratory for cultivation.

Examples of transport media include:Thioglycolate broth for strict anaerobes.

The colonial appearance on culture media:

Shape:The colonies may be small (pin-point) fimbriate, flat or convex.

Colour:• The colonies may be colourless or bacteria produce endopigments which give the colonies a characterestic colour , e.g. Staph. aureus produce golden yellow colonies. Staph. albus produce white endopigment. Staph. Citreus produce a lemon yellow endopigment.• The bacteria may produce exopigments,e.g. Pseudomonas aeruginosa produce a green exopigment in the surrounding media.

Antimicrobial Chemotherapy:An antibacterial agent is a compound or substance that kills or slows down the growth of bacteria.Antibiotic(s) has come to include a broader range of antimicrobial compounds, including anti-fungal and other compounds.It is produced by microbes and is harmful to other microbes, except viruses.These include, for example, the beta-lactam antibacterials, which include the penicillins (produced by fungi in the genus Penicillium notatum),and the cephalosporins.

Compounds that are still isolated from living organisms are the aminoglycosides, whereas other chemotherapeutic agents—for example, the sulfonamides,and the quinolones, are produced by chemical synthesis.

Classification of Antibiotics:

According to agent action: In this classification antibacterial agents are divided into two broad groups according to their biological effect on microorganisms: bactericidal agents kill bacteria, and bacteriostatic agents slow down or stall bacterial growth.

Bactericidal antibiotics: Antibiotics that inhibit cell wall synthesis: the Beta-lactam antibiotics (penicillin derivatives, and cephalosporins). Aminoglycosidic antibiotics are usually considered bactericidal, although they may be bacteriostatic with some organisms.

Bacteriostatic antibiotics limit the growth of bacteria by interfering with bacterial protein production, DNA replication, or other aspects of bacterial cellular metabolism.

This group includes:Tetracyclines, sulphonamides , trimethoprim ,chloramphenicol, and macrolides.

Antibiotic sensitivity test:

Antibiotic sensitivity is a term used to describe the susceptibility of bacteria to antibiotics. Antibiotic susceptibility testing (AST) is usually carried out to determine which antibiotic will be most successful in treating a bacterial infection in vivo. Testing for antibiotic sensitivity is often done by the Kirby-Bauer method ( Disc-diffusion method).

Other methods to test antimicrobial susceptibility include the  E-test (also based on antibiotic diffusion).

Agar and Broth dilution methods for Minimum Inhibitory Concentration determination.

Antibiotic sensitivity test:

Antibiotic sensitivity Test : Different antibiotics have been placed on an agar plate growing bacteria and the plate is incubated. The degree of inhibition of growth around the discs is measured.The more the zone of inhibition, the more the sensitivity to the antibiotics

The Dilution Method:

Serial dilutions of antibiotics are incorporated in agar containing or broth culture media.

The lowest concentration of antibiotic that prevents visible growth after an 18-24 hours incubation period is known as minimal inhibitory concentration (MIC).

The minimal bactericidal concentration (MBC) may be determined in broth dilution tests by subculturing the containers that show no growth on to antibiotic-free agar containing media.

The lowest concentration of antibiotic that totally suppresses growth after overnight incubation is known as MBC.