Biochemical Activities of Microorganisms Part (1) Microbiology Lab (8) Biochemical Activities of Microorganisms Part (1) Abdelraheem BA

Purpose of the lab To understand how enzymatic activity can be used to identify a microorganism. To understand how each enzyme works. To perform and interpret a series of biochemical assays useful for bacterial identification.

Terms to know Observation Vs Interpretation. Observation; The result of the test. Interpretation; What does that result indicate.

Medical microbiology processing Sample collection. Urine, blood, CSF, vaginal swab, skin, sputum… etc. Isolation. Streak plate with sample. Purification. Subculture to isolate pure colonies Identification. Next slide

Identification Phynotyping identification: Genotyping identification: Microscopic morphology. Simple & differential stains. Cultural characteristics. Biotyping. Enzymatic activity. Serotyping. Ag-Ab reactions (O & H antigens) Antibiotyping. Antibiotic sensitivity tests. Genotyping identification: DNA probes and PCR. Identification of MO at the molecular level.

Biotyping Everything that a living MO does is a result of enzymatic activity. The sum of enzymatic activities of a MO is its Biochemical fingerprint. Biotyping, is the test of enzymatic activity.

Enzymes Exoenzymes-Extracellular enzymes: Starch hydrolysis (Amylase). Lipid hydrolysis (Lipase). Casein hydrolysis (Protease). Gelatin hydrolysis (Gelatinase). Blood hemolysis (Streptolysin) Endoenzymes-Intracellular enzymes: Catalase. Oxidase. IMViC (Indole, Methyl red,, Voges Poskauer & Citrate) enzymes. Nitrate reduction enzymes. Urease. Carbohydrate fermentation enzymes.

Blood agar, Mannitol Salt agar & MacConkey agar Please, go to lab (5)

Catalase Function: How does it work? Most bacteria, which grow aerobically, produce hydrogen peroxide H2O2 as a by-product of respiration. Some bacteria have the ability to neutralize the toxic effect of H2O2 by Catalase. How does it work? 2H2O2 2H2O + O2(g) Liberation of gas result in bubbles. Catalase

Purpose TAKE NOTES. To differentiate between genera: Streptococcus Vs Staphylococcus and Micrococcus. Clostridium Vs Bacillus.

Procedure Slide method: Pick the center of an 18-24 hours pure colony with an inoculating needle. Place it on a clean glass slide. Add a drop of 30% H2O2 over the organism on the slide. Observe immediate bubbling and record result.

Procedure Tube method: Directly add 1 ml of 30% H2O2 to an 18-24 hours heavily inoculated pure agar slant tube. DON’T USE A BLOOD AGAR MEDIA. Observe for immediate bubbling.

Results

Precautions H2O2 must be fresh and must be kept away from light. Don’t use blood agar in this test. False positive results, why? During procedure don’t reverse the order. Meaning that: addition of 30% H2O2 then using the needle to add bacteria. Due to the reaction of platinum with H2O2 . Old colonies may lose their catalase activity. False negative results. Avoid exposure of H2O2 to skin. H2O2 must be fresh and must be kept away from light.

Coagulase It is considered as a virulence factor. This enzyme enables bacteria to clot the plasma, which allows these bacteria to evade host’s immune system. Used to differentiate between species within the genera Staphylococcus. S. aureus, is coagulase positive. S. epidermitis & S. saprophaticus are coagulase negative. It is usually the final diagnosis criterion of Staphylococcus identification. How? TAKE NOTES

Procedure Slide method – to detect BOUND Coagulase. Place one drop of plasma in the center of a clean slide. Transfer a large amount of bacterial growth into the plasma. Mix well, until you get a homogenous suspension. Results should be taken within 10-15 seconds. NEGATIVE CONTROL (Normal saline & bacteria). To exclude auto-coagulation.

Procedure Tube method – used to detect free & bound coagulases. Place 0.5 ml of sterile plasma into a clean tube. Place 0.5 ml of bacterial broth or two loopfuls from solid culture. Mix the tube. Incubate the test tube for 4 hours at 35ºC. Results should be taken every 30 minutes. After 4 hours, if the result was negative, reincubate over night.

Results You don’t report the results as Coagulase positive, until you make sure that the negative control is actually negative!

Oxidase Is a Cytochrome oxidase. Found in bacteria that transfers electrons to oxygen. This enzyme oxidizes reduced Cyt C to make this transfer of energy. Detection of oxidase presence is done using an Oxidase disk.

Purpose Used to identify Neisseria species. Used to separate Pseudomonadaceae family form oxidase negative members of Enterobacteriaceae. Aid in differentiation between genera: Moraxella (+) Neisseria (+) Acinetobacter (-)

Procedure Pick up an isolated colony. Transfer it to a filter paper strip, using a wooden stick. This paper is impregnated with: N.N.N.N Tetraethyl paraphenylen Diamino Dihydrochloride (TPD).

Results Purple No color change Positive. Examples: Pseudomonas spp., Neisseria spp. No color change Negative. Examples: Enterobacteriaceae, Acinetobacter.