Adigun hafsat oluwabusola
19/MHS06/031
Medical laboratory science
MCB 202
Assignment
QUESTION
 1) Explain (step by step) at least ten (10) bichemical reactions of bacteria.
2) Explain the identification/ staining techniques of fungi.
 Answer( 1)
 I) OXIDASE TEST;
    The oxidase test is a test used  to determine if a bacterium produces certain cytochrome c oxidases. It uses disks impregnated with a reagent such as N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) or N,N-dimethyl-p-phenylenediamine (DMPD), which is also a redox indicator. The reagent is a dark-blue to maroon color when oxidized, and colorless when reduced. Oxidase-positive bacteria possess cytochrome oxidase or indophenol oxidase (an iron-containing hemoprotein).These both catalyze the transport of electrons from donor compounds (NADH) to electron acceptors (usually oxygen). The test reagent, TMPD dihydrochloride acts as an artificial electron donor for the enzyme oxidase. The oxidized reagent forms the colored compound indophenol blue. The cytochrome system is usually only present in aerobic organisms that are capable of using oxygen as the terminal electron acceptor. The end-product of this metabolism is either water or hydrogen peroxide (broken down by catalase).
Strains may be either oxidase-positive (OX+) or oxidase-negative (OX-).
OX+
OX+ normally means the bacterium contains cytochrome c oxidase and can therefore use oxygen for energy production by converting O2 to H2O2 or H2O with an electron transfer chain.
The Pseudomonadaceae are typically OX+
The Gram-negative diplococci Neisseria and Moraxella are oxidase-positive.
Many Gram-negative, spiral curved rods are also oxidase-positive, which includes Helicobacter pylori, Vibrio cholerae, and Campylobacter jejuni.
Oxidase Variable
Legionella pneumophila may be oxidase-positive.
OX−
OX− normally means the bacterium does not contain cytochrome c oxidase and, therefore, either cannot use oxygen for energy production with an electron transfer chain or employs a different cytochrome for transferring electrons to oxygen.
Enterobacteriaceae are typically OX−.

II) CATALASE TEST
The enzyme catalase mediates the breakdown of hydrogen peroxide into oxygen and water. The presence of the enzyme in a bacterial isolate is evident when a small inoculum is introduced into hydrogen peroxide, and the rapid elaboration of oxygen bubbles occurs. The lack of catalase is evident by a lack of or weak bubble production. The culture should not be more than 24 hours old.
Bacteria thereby protect themselves from the lethal effect of Hydrogen peroxide which is accumulated as an end product of aerobic carbohydrate metabolism.
                                 2H2O2 ----------> 2H2O + O2 (gas bubbles)
 Uses of Catalase Test
*The morphologically similar Enterococcusor Streptococcus (catalase negative) and Staphylococcus (catalase positive) can be differentiated using the catalase test.
*Also valuable in differentiating aerobic and obligate anaerobic bacteria.
*Semiquantitative catalase test is used for the identification of Mycobacterium tuberculosis.
*It is used to differentiate aerotolerant strains of Clostridium, which are catalase negative, from Bacillus species, which are positive.
*Catalase test can be used as an aid to the identification of Enterobacteriaceae.
INTERPRETATION OF TEST
Positive: Copious bubbles produced, active bubbling
Examples: Staphylococci, Micrococci, Listeria, Corynebacterium diphtheriae, Burkholderia cepacia, Nocardia, the family Enterobacteriaceae (Citrobacter, E. coli, Enterobacter, Klebsiella, Shigella, Yersinia, Proteus, Salmonella, Serratia), Pseudomonas, Mycobacterium tuberculosis, Aspergillus, Cryptococcus, and Rhodococcus equi.

Negative: No or very few bubbles produced.
Examples: Streptococcus and Enterococcus spp

III) SUGAR FERMENTATION;
When microorganisms ferment carbohydrate an acid or acid with gas are produced. Depending upon the organisms involved and the substrate being fermented, the end products may vary. Common end-products of bacterial fermentation include lactic acid, formic acid, acetic acid, butyric acid, butyl alcohol, acetone, ethyl alcohol, carbon dioxide, and hydrogen.
The production of the acid lowers the pH of the test medium, which is detected by the color change of the pH indicator. Color change only occurs when sufficient amount of acid is produced, as bacteria may utilize the peptone producing alkaline by-products.
Phenol red is commonly used as a pH indicator in carbohydrate fermentation tests. Other pH indicators such as bromocresol/bromocresol purple (BCP), bromothymol/bromothymol blue (BTB), and Andrade’s can be used.
USES OF SUGAR FERMENTATION TEST
sugar fermentation patterns can be used to differentiate among bacterial groups or species.
1)All members of Enterobacteriaceae family are glucose fermenters (they can metabolize glucose anaerobically).
2)Maltose fermentation differentiates Proteus vulgaris (positive) from Proteus mirabilis (negative).
3)Both Neisseria gonorrhoeae (gonococci) and Neisseria meningitides (meningococci) ferments glucose, but only meningococci ferments maltose.
4)Rapid carbohydrate utilization tests (RCUTs) can be performed to identity Corynebacterium diphtheriae and other Corynebacterium species.
TEST INTERPRETATION
Acid production:
Positive: After incubation the liquid in the tube turns yellow (indicated by the change in the color of the phenol red indicator). It indicates that there is drop in the pH because of the production of the acid by the fermentation of the carbohydrate (sugar) present in the media.
Negative: The tube containing medium will remain red, indicating the bacteria cannot ferment that particular carbohydrate source present in the media.
Gas Production
Positive: A bubble (small or big depending up the amount of gas produced) will be seen in the inverted Durham tube.
Negative: There won’t be any bubble in the inverted Durham tube i.e. bacteria does not produce gas from the fermentation of that particular carbohydrate present in the media i.e. anaerogenic organisms.

IV) COAGULASE TEST;
Coagulase is an enzyme-like protein and causes plasma to clot by converting fibrinogen to fibrin. Staphylococcus aureus produces two forms of coagulase: bound and free. 

Bound coagulase (clumping factor) is bound to the bacterial cell wall and reacts directly with fibrinogen. This results in an alternation of fibrinogen so that it precipitates on the staphylococcal cell, causing the cells to clump when a bacterial suspension is mixed with plasma. This doesn’t require coagulase-reacting factor.

Free coagulase involves the activation of plasma coagulase-reacting factor (CRP), which is a modified or derived thrombin molecule, to from a coagulase-CRP complex. This complex in turn reacts with fibrinogen to produce the fibrin clot.
INTERPRETAION OF TEST
i)Clumping in both drops of slides indicates that the organism auto agglutinates and is unsuitable for the slide coagulase test. All the negative slide test must be confirmed using the tube test. 
ii)During slide test, there may be chance to false positive results in case of citrate utilizing bacteria ( Enterococcus and Pseudomonas). In this case also, tube test should be performed and confirmed.
Examples
Coagulase Positive Organisms: Staphylococcus aureus and other animal host bacteria like  S. pseudintermedius, S. intermedius, S. schleiferi,  S. delphini, S. hyicus, S. lutrae, S. hyicus
Coagulase Negative Organisms: Staphylococcus epidermidis, S. saprophyticus, S. warneri, S. hominis, S. caprae, etc.
V) IMVC TEST;
Each of the letters in “IMViC” stands for one of these tests. “I” is for indole; “M” is for methyl red; “V” is for Voges-Proskauer, and “C” is for citrate,  lowercase “i” is added for the ease of pronunciation. IMViC is an acronym that stands for four different tests
1)Indole test
2)Methyl red test
3)Voges-Proskauer test
4)Citrate utilization test
To obtain the results of these four tests, three test tubes are inoculated: tryptone broth (indole test), methyl red – Voges Proskauer broth (MR-VP broth), and citrate. IMViC tests are employed in the identification/differentiation of members of family enterobacteriaceae.
1) INDOLE TEST
It is performed on sulfide-indole-motility (SIM) medium or in tryptophan broth, or in motility urease indole (MIU) medium. Result is read after adding Kovac’s reagent.
i) The positive result is indicated by the red layer at the top of the tube after the addition of Kovács reagent.
ii) A negative result is indicated by the lack of color change at the top of the tube after the addition of Kovács reagent.
Methyl red test and Voges-Proskauer test both are done in methyl red–Voges-Proskauer (MR-VP) broth, but the reagents that are added varies according to the test.
2) METHYL RED (MR) TEST
i) Positive methyl red test are indicated by the development of red color after the addition of methyl red reagent.
ii) A negative methyl red test is indicated by no color change after the addition of methyl red reagent
3) VOGES-PROSKAUER (VP) TEST;
i)Negative test is indicated by lack of color change after the addition of Barritt’s A and Barritt’s B reagents.
ii)A positive Voges-Proskauer test is indicated by the development of red-brown color after the addition of Barritt’s A and Barritt’s B reagents.
4) CITRATE UTILIZATION TEST
The test is performed on Simmons citrate agar:

i) Negative citrate utilization test is indicated by the lack of growth and color change in the tube
 ii) A positive citrate result as indicated by growth and a blue color change. 

IMViC Test results of Some Genera of Enterobacteriaceae:       

1)IMViC tests of Escherichia coli
i)Indole: Positive
ii)Methyl-Red: Positive
iii)Voges-Proskauer test: Negative
iv)Citrate test: Negative

6) STARCH HYDROLYSIS TEST;
This test is used to identify bacteria that can hydrolyze starch (amylose and amylopectin) using the enzymes a-amylase and oligo-1,6-glucosidase. Often used to differentiate species from the genera Clostridium and Bacillus. Because of the large size of amylose and amylopectin molecules, these organisms can not pass through the bacterial cell wall. In order to use these starches as a carbon source, bacteria must secrete a-amylase and oligo-1,6-glucosidase into the extracellular space. These enzymes break the starch molecules into smaller glucose subunits which can then enter directly into the glycolytic pathway. In order to interpret the results of the starch hydrolysis test, iodine must be added to the agar. The iodine reacts with the starch to form a dark brown color. Thus, hydrolysis of the starch will create a clear zone around the bacterial growth.

7) GELATIN HYDROLYSIS TEST;
   Gelatin hydrolysis test is used to detect the ability of an organism to produce gelatinase (proteolytic enzyme) that liquefy gelatin.  Hydrolysis of gelatin indicates the presence of gelatinases.
This process takes place in two sequential reactions. In the first reaction, gelatinases degrade gelatin to polypeptides. Then, the polypeptides are further converted into amino acids.  The bacterial cells can then take up these amino acids and use them in their metabolic processes.
Gelatin hydrolysis test is helpful in identifying and differentiating species of Bacillus, Clostridium, Proteus, Pseudomonas, and Serratia.
It distinguishes the gelatinase-positive, pathogenic Staphylococcus aureus from the gelatinase-negative, non-pathogenic S. epidermidis . Gram-positive, spore-forming, rodshaped, aerobic or anaerobic bacteria such as Bacillus anthracis, Bacillus cereus, Bacillus subtilis, Clostridium perfringens and Clostridium tetani, are also positive for gelatin hydrolysis.
The test can also be used to differentiate genera of gelatinase-producing bacteria such Serratia and Proteus from other members of the family Enterobacteriaceae.
Expected results
Positive: Partial or total liquefaction of the inoculated tube (uninoculated control medium must be completely solidified) even after exposure to cold temperature of ice bath or refrigerator (4°C)

Gelatin Hydrolysis Test: Above tube: Positive Below Tube: Negative
Gelatin Hydrolysis Test: Above tube: Positive
Below Tube: Negative
Negative: Complete solidification of the inoculated tube even after exposure to cold temperature of ice bath or refrigerator (4°C)

Common bacteria and their reactions to the gelatin hydrolysis test performed on nutrient gelatin.

8) UREASE TEST;
Urea is a diamide of carbonic acid.  It is hydrolyzed with the release of ammonia and carbon dioxide. Many organisms especially those that infect the urinary tract, have an urease enzyme which is able to split urea in the presence of water to release ammonia and carbon dioxide.
The ammonia combines with carbon dioxide and water to form ammonium carbonate which turns the medium alkaline, turning the indicator phenol red from its original orange yellow color to bright pink.
                                                 urease
              (NH2)2CO + 2 H2O ------------->   CO2      +        H2O   +   2 NH3
               Urea                                            carbon dioxide    water       ammonia
Result and Interpretation 
Organisms that hydrolyze urea rapidly (Proteus spp., Morganella morganii, and some Providencia stuartii strains) will produce strong positive reactions within 1 or 6 hours of incubation;  delayed positive organisms (e.g. Klebsiella spp and Enterobacter species ) will produce weak positive reactions in the slant in 6 hours of incubation which will be intense during further incubation. The culture medium will remain a yellowish color if the organism is urease negative e.g. Escherichia coli. 

9) NITRATE PRODUCTION TEST;
Nitrate reduction test is used for the differentiation of members of Enterobacteriaceae on the basis of their ability to produce nitrate reductase enzyme that hydrolyze nitrate (NO3–) to nitrite (NO2–) which may then again be degraded to various nitrogen products like nitrogen oxide, nitrous oxide and ammonia (NH3) depending on the enzyme system of the organism and the atmosphere in which it is growing.
Other uses of Nitrate Reduction Tests are: 
                                                                             Nitrate  (NO3)
                                                                                      |
                                                                             nitrate reductase
                                                                                      |
                                                                              Nitrite (NO2)
                                                                                      |
                                                                              nitrite reductase
                                                                                      |
                                                                              Nitric oxide (NO)
                                                                                      |
                                                                              nitric oxide reductase
                                                                                      |
                                                                              Nitrous oxide (N2O)
                                                                                      |
                                                                              nitrous oxide reductase
                                                                                      |
                                                                               Nitrogen (N2)
                                               NITRATE REDUCTION PATHWAY
i) Differentiating Mycobacterium species.
ii) Identifying species of Neisseria and separating them from Moraxella and Kingella species. The nitrate reduction test is a critical test for differentiating between N. gonorrhoeae and K. denitrificans, particularly when strains of K. denitrificans appear to be gram-negative diplococci in stained smears.
iii) Facilitating species identification of Corynebacterium

Result and Interpretation
i) Nitrate Reduction Positive:  (Red after sulfanilic acid + alpha-naphthylamine; no color after zinc)
ii) Nitrate Reduction Negative:  (No color after sulfanilic acid + alpha-naphthylamine followed by Red after zinc)

10) HYDROGEN SULPHIDE PRODUCTION TEST;
Hydrogen sulphide (H2S) production test is used for the detection of hydrogen sulphide (H2S) gas produced by an organism. It is used mainly to assist in the identification of members of family Enterobacteriaceae. H2S is produced by certain bacteria through reduction of sulphur containing amino acids like cystine, methionine or through the reduction of inorganic sulphur compounds such as thiosulfates, sulfates or sulfites. The hydrogen sulphide production can be detected by incorporating a heavy metal salt containing iron or lead as H2S indicator to a nutrient culture medium containing cystine and sodium thiosulfates as the sulfur substrates. Hydrogen sulphide, a colorless gas, if produced reacts with the metal salt forming visible insoluble black precipitate of ferrous sulphide.
Interpretation of test;
Positive result: blackening on the medium
H2S+ve
Negative result: no blackening on the medium
H2S-ve.

ANSWER (2)
The identification of fungi is made using combination of
i) Growth rate
ii) Colonial morphological feature
iii) Microscopic observation
1. Growth rate
i) Growth rate is one of the helpful observation made when examining fungi. However this information may be of limited value since the growth rate of certain fungi is variable depending upon the inoculum.
ii) In general growth rate of dimorphic fungi such as Blastomyces dermatitidis, Histoplasma capsulatum is 1-4 weeks.
iii) Colonies of Zygomycetes appears within 24 hours
 iv) Similarly, Saccharomyces give visible colonies in 3-5 days
2. Colonial morphological feature:
i) Colony morphology is one of the important feature of fungi but still it has limited value in identification of mold because of natural variation among isolates and variation of colonies on different culture media.
ii)When examining the colonial morphological feature of fungi, types of culture media used and incubation condition should be considered. For example Histoplasma capsulatum appears as a white mold on BHI agar and appears as yeast when grown on same media containing blood enrichment.
iii) Colonial morphology can be used to supplement the information obtained from microscopic examination.
3. Microscopic observation:
i) In general, microscopic morphological feature of fungi are stable and exhibit minimal variation.
ii) The definite identification is based on characteristics, shape, methods of production and arrangement of spores. However, the size of hyphae also provides helpful information identification of fungi.
iii) For microscopic examination, stained preparation is performed.
Common used staining reagent in microscopic observation are;
i) Lactophenol cotton blue: stains cytoplasm
ii) Cresyl blue stain: stains spore wall of basidiomycetes
iii) Periodic acid Schiff (PAS) stain: distinguish hyphae from other fungal elements
iv) Hematoxylin stain: stains nucleus in filamentous fungi
v) Giemsa stain: stains nucleus in filamentous fungi
vi) Calcofluor white stain
vii) India ink
vii) Wright stain
I) LACTOPHENOL COTTON BLUE;
The lactophenol cotton blue (LPCB) wet mount preparation is the most widely used method of staining and observing fungi and is simple to prepare. The preparation has three components:
1) Phenol:   kills any live organisms;
2) Lactic acid : It  preserves fungal structures, and
3) Cotton blue : It stains the chitin in the fungal cell walls.
Lactophenol Cotton Blue Solution is a mounting medium and staining agent used in the preparation of slides for microscopic examination of fungi. Fungal elements are stained intensely blue