Basic Microbiology Laboratory for Dental Students M-265 By Lecturer Yasemin Al-Shboul Spring 2011-2012

Physical and Chemical Agents That Affect Microbial Growth

Antimicrobial Agent: Chemical substance used in treatment of infectious diseases. Antimicrobial Agents (According to activity) are either Bateriostatic (Inhibits bacterial growth) or Bacteriocidal (Kills bacteria). Types of Antimicrobial Agents: 1- Antiseptics: Bactericidal agents, can be used on skin and mucus membranes. Not for internal use. E.g. Alcohol, Iodine solution, Detergents, Mercurials, Silver nitrate. 2- Disinfectants: Bactericidal agents, only used for inanimate objects. E.g. Chlorine, Hypochlorites, Chlorine compounds, Copper sulfate, Quaternary ammonium compounds. - Sterilization kills all forms of life (including spores), but Antiseptics and Disinfectants my not necessarily kill spores. -Safety of a compound depends on concentration. 3- Chemotherapeutic Agents (Synthetic Drugs): Antimicrobial agents of synthetic origin useful in the treatment of microbial or viral diseases. E.g. Sulfonamides, Isonazide, Ethambutol, AZT, Chloramphenicol. Note: Agents used for treatment of diseases of non-microbial origin are not considered as chemotherapeutic agents. 4- Antibiotics: Antimicrobial agents produced by microorganisms that kill or inhibit the growth of other microorganisms. E.g. Penicillin.

Kirby Bauer Method

1- Disk Diffusion Method Also Known as the Standard Disk Susceptibility Test. Procedure: Inoculate Agar plate with a standard inoculum, place Disks, Incubate, measure diameter of Inhibition zone (if any), compare with standard tables. Zone of Inhibition Depends on: a. Amount of antimicrobial agent. b.Solubility of the agent. c. The Diffusion coefficient. d.The overall effectiveness of the agent. e.Type of microorganisms. f. Type of Media: Muller Hinton media must be used. g. Agar Depth: Must be 4mm. h.Type and Concentration of the Antibiotic Used. I. Number of Bacteria: Must be 1-1.5 * 108 cells/ml. J. pH of Media.

 Dilution Methods:
Used to determine the MIC and MBC of the Antimicrobial Agent. MIC (Minimum Inhibitory concentration): Lowest concentration of the antimicrobial agent required to inhibit the growth of a bacterial isolate. MBC (Minimum Bactericidal Concentration): Lowest Concentration of the antimicrobial agent required to kill the bacterial isolate.

A) Tube Method:
Serial dilution of the antibiotic in Broth: 0.1 to 128mg/ml Measures the MIC and the MBC of the antibiotic. Procedure.

 - Standard Inoculum = 1.5 * 108 cells/ml, similar in turbidity to a 0.5 McFarland standard.
McFarland standards are used as a reference to adjust the turbidity of bacterial suspensions so that the number of bacteria will be within a given range .

Kirby bauer test:To observe the efficacy of various antibiotic agents against a range of gram positive and gram negative Moneran strains through measurement of zones of inhibition

Biochemical Activities of Microorganisms

Medical Microbiology Processing:

Collection (Transport Media): Urine, blood, stool, vaginal swab, skin, throat, and sputum. Isolation (Selective Media): Streak plate, pour plate, and spread plate. Purification: Subculture for isolation of a pure colony

Phenotyping:
Microscopic Morphology. Cultural characteristics. Biotyping. Stereotyping (Ag-Ab reactions): O-Ag, H-Ag Antibiotyping.

Genotyping: DNA probes, PCR.

Biotyping - Everything that a living organism does is the result of the activity of an enzyme.
- The sum of the activities of all an organisms enzymes equals its Biochemical Fingerprint. - An organism is the totality of its enzymes and their products, so by determining which enzymes are present, one can describe and identify that organism.: Test of enzymatic activity. Types of Enzymes:

Extracellular enzymes:
Amylase: Starch hydrolysis. Lipase: Lipid hydrolysis. Protease: Casein hydrolysis. Gelatinase: Gelatin hydrolysis. Streptolysin: Blood hydrolysis.

Intracellular enzymes:
Catalase. Oxidase. IMViC. Nitrate Reduction enzymes. Urease Carbohydrate Fermentation enzymes

Blood Agar: Enriched, differential medium, for the isolation of fastidious microorganisms e.g. streptococcus spp. Sheep, Human, or Rabbit Blood Agar. Blood Concentration 5-10%. Hemolysis pattern: Beta hemolysis (): Complete hemolysis of hemoglobin e.g. Streptococcus pyogenes. Alpha hemolysis (): Incomplete lysis of RBCs with reduction of hemoglobin to methemoglobin, resulting in a greenish color around the bacterial colony e.g. Streptococcus pneumoniae. Gamma hemolysis (): no lysis of RBCs & no change in the medium e.g. Stretococcus fecalis. Manitol Salt Agar (MSA) Selective, differential medium for the isolation of Staphylococcus spp. Components: 7.5% NaCl, Manitol, Phenol Red (pH indicator). MacConkey Agar: Selective, differential Medium Inhibitors (Bile salts & Crystal violet): Prevent growth of Gram positive bacteria. Lactose & Neutral Red (pH indicator): differentiates between Lactose(Pink) and non-Lactose (colorless) fermenters.

 Blood agar hemolytic reactions

 MacConkeys agar

 Mannitol Salt Agar

:Catalase Test
Most organisms that grow aerobically produce hydrogen peroxide (H2O2). H2O2 is toxic to cells and must be converted to non-toxic compounds in order for the cell to survive. H2O2 + Catalase H2O + O2 (Bubbles) E.g. Staph.: Catalase +ve, Strep.: Catalase ve Blood agar will give a False Positive result. The bubbles resulting from production of oxygen gas clearly indicate a catalase positive result. The sample on the right below is catalase positive. The Staphylococcus spp. and the Micrococcus spp. are catalase positive . The Streptococcus and Enterococcus spp. are catalase negative.

Coagulase Test
Coagulase test is used to differentiate between Staphylococcus aureus and other coagulase negative species. This test is based on the coagulative properties of S. aureus with plasma. S. aureus produces 2 forms of coagulase free and bound. Different types of tests are used to observe the coagulation reactions, depending on the form of coagulase targeted. Bound coagulase, also known as clumping factor, is detected using the slide test. This form of coagulase is bound to the bacterial cell wall, hence its name. It reacts with fibrinogen in plasma to form visible clumps. However this test is not performed in the lab anymore. The tube test is done on free coagulase. Free coagulase released by the bacterial cell is often found in culture filtrates. It exists as a free form, unlike the bound coagulase attached to the cell. The free coagulase acts on prothrombin, producing a complex with similar properties to thrombin - staphylothrombin. This substance then acts on fibrinogen to form a fibrin clot in plasma.

2-5 staphylococcal colonies are suspended in 0.5ml of rabbit plasma w/EDTA. Incubate aerobically at 35oC for 4 hours,check every 30 min. Check for clot formation. If negative, continue incubation overnight (12 hours)and check again after the extended incubation.

 Goagulase test

Oxidase Test
Cytochrome Oxidase is an enzyme (which is a redox indicator) found in some bacteria that transfers electrons from (reduced Cytochrome C) to Oxygen the final electron acceptor in some electron transport chains. In the test the Oxidase disk (N,N,N,N Tetraeythyl paraphenylen diamino dihydrochloride) acts as an electron donor to cytocrome oxidase. If the bacteria oxidizes the disk (removes electrons), then the disk or filter paper will turn purple, indicating a positive test. E.g. Pseudomonas Spp. And Neisseria spp.. No color change indicates a negative test e.g. Enterobacteriaceae.

 All images are for oxidase test results

GOOD LUCK FOR EACH OF YOU .