Biochemical Activities of Microorganisms Part (1)
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Microbiology Lab (8)Biochemical Activities of Microorganisms
Part (1)
Abdelraheem BA
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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.
Purpose of the lab
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Observation Vs Interpretation.◦ Observation;
The result of the test.◦ Interpretation;
What does that result indicate.
Terms to know
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Sample collection.◦ Urine, blood, CSF, vaginal swab, skin, sputum…
etc. Isolation.
◦ Streak plate with sample. Purification.
◦ Subculture to isolate pure colonies Identification.
◦ Next slide
Medical microbiology processing
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Phynotyping 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.
Identification
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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.
Biotyping
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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.
Enzymes
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Please, go to lab (5)
Blood agar, Mannitol Salt agar & MacConkey agar
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Function:◦ 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
Catalase
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TAKE NOTES. To differentiate between genera:
◦ Streptococcus Vs Staphylococcus and Micrococcus.
◦ Clostridium Vs Bacillus.
Purpose
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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
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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.
Procedure
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Results
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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.◦ False positive results, why?
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.
Precautions
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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
Coagulase
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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
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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.
Procedure
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Results
You don’t report the results as Coagulase positive, until you make sure that the negative control is actually negative!
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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.
Oxidase
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Used to identify Neisseria species. Used to separate Pseudomonadaceae family
form oxidase negative members of Enterobacteriaceae.
Aid in differentiation between genera:◦ Moraxella (+)◦ Neisseria (+)◦ Acinetobacter (-)
Purpose
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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).
Procedure
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Purple◦ Positive.◦ Examples: Pseudomonas spp., Neisseria spp.
No color change◦ Negative.◦ Examples: Enterobacteriaceae, Acinetobacter.
Results