Materials and Methods
3. CO 2 evolution by Soda-Lime method (Kalamdhad et al., 2008)
3.3 PHASE II: Microbial Studies on the Best Trial of Water Hyacinth Compost
3.3.2 Culture Dependent Bacterial Identification
3.3.2.2 Biochemical Tests
1. Gram Staining: For gram staining Himedia K004-1KT capsules satin kit was used.
S025 Nigrosin stains 10% w/v 100 mL. S021 Methylene blue 125 mL. A loopful of culture was placed on glass slide and stained with 10% w/v Nigrosin stain (S025).
The smear was mixed well and allowed to air dry and then fixed with gentle heat.
Thereafter, the smear was stained with the methylene blue (S021) for 30 s. Rinsed rapidly with water and air dried. The glass slide was observed under the immersion oil lens. Gram positive bacteria: Stained dark purple colour due to retaining the primary dye called crystal Violet in the cell wall. Gram negative bacteria: Stained pink due to retaining the counter staining dye called safranin.
2. Capsule stain: For capsule detection, capsule stains kit (K004) was used. As smear of microbial culture was prepared on a glass slide. Drying and heat fixation was done with gentle heating. The slide with bacterial film on upper side was kept over a beaker with boiling water. When large droplets condensed on the lower side of the slide the smear was flooded with Schaeffer and Fulton spore stain A (S028). Finally the slide
was steamed for 3-6 min and rinsed under running tap water. Counter staining with Schaeffer and Fulton spore stain B (S029) was done for 30 s. The slide was washed with water, dried and observed under immersion oil lens.
3. Spore staining: Schaeffer and Fulton’s spore stain kit K006-1Kt (Himedia) was used for detection of spores in the microbial culture. Following steps were followed for the process: Smear microbial culture on a glass slide and heat fix, cover it with a square of blotting paper. Saturate the blotting paper with carbolfuchsin and steam for 5-10 min, keep the paper moist and add 2 drops dye as required. Alternatively, the slides may be steamed over a container of boiling water. Remove the blotting paper and decolorize the film with acid-alcohol for 1 min, rinse with tap water and blot dry. Dry a thin even film of saturated aqueous nigrosin on the slide. Examine the slide under the oil immersion lens (1,000×) for the presence of endospores. Vegetative cells are colourless, endospores are red, and the background is black.
4. Catalase test: The catalase enzyme serves to neutralize the bactericidal effects of hydrogen peroxide. Catalase expedites the breakdown of hydrogen peroxide (H2O2) into water and oxygen. (2 H2O2+ Catalase−−→2 H2O + O2). This reaction is evident by the rapid formation of bubbles. There are many applications and method variations of the catalase test. These include the slide or drop catalase test, the tube method, the semi quantitative catalase for the identification ofMycobacterium tuberculosis, the heat-stable catalase used for the differentiation of Mycobacterium species, and the capillary tube and cover slip method. The following steps were followed for the test.
Place a microscope slide inside a petri dish. Keep the petri dish cover available. Use a sterile inoculating loop collected a small amount of organism from a well-isolated 18- to 24 h colony and placed it onto the microscope slide. This is particularly important if the colony isolate was grown on agar containing red blood cells. Carryover of red blood cells into the test may result in a false-positive reaction. Use a dropper placed 1 drop of 3% H2O2onto the organism on the microscope slide.. Immediately cover the petri dish with a lid to limit aerosols and observe for immediate bubble formation (O2 + water = bubbles). Observe the formation of bubbles against a dark background to enhance readability. Positive reactions are evident by immediate effervescence (bub- ble formation). Place microscope slide over a dark background and use a magnifying glass of microscope and observe weak positive reactions. Use a microscope, placed a cover slip over the slide and view under 40×magnifications.
5. Oxidase test: Oxidase test using N, N,N,N-tetramethyl-p-phenylenediamine reagent (Himedia GRM445), Kovács oxidase reagent (1%), tetra-methyl-p-phenylenediamine
3.3. PHASE II: Microbial Studies on the Best Trial of Water Hyacinth Compost
dihydrochloride, in water was performed. The oxidase test is a biochemical reac- tion that assays for the presence of cytochrome oxidase, an enzyme sometimes called indophenol oxidase. In the presence of an organism that contains the cytochrome ox- idase enzyme, the reduced colourless reagent becomes an oxidized coloured product.
6. Filter spot method: Following steps were followed for the test: Pick a microbial colony (18 to 24 h culture) by a loop and rub onto a small piece of filter paper. Place 2 drops of 1% Kovács oxidase reagent on the microorganism smear. Observe colour changes. Bacteria show dark purple within 5 to 10 s oxidase positive. Microorganisms are delayed oxidase positive when the colour changes to purple within 60 to 90 s.
Microorganisms are oxidase negative if the colour does not change or it takes longer than 2 min. Motility test For motility test Mannitol Motility Test medium (Himedia M770) was used: Use a sterile needle to pick a well-isolated colony and stab the medium to within 1 cm of the bottom of the tube. Be sure to keep the needle in the same line as it entered as it is removed from the medium. Incubate at 35◦C for 18 h or until growth is evident. A positive motility test is indicated by a diffuse cloud of growth away from the line of inoculation.
7. Voges-Proskauer Test:Barrett’s reagent A: 5% (w/v) a-naphthol in absolute ethanol.
Barrett’s reagent B: 40% (w/v) KOH in deionized water (this might be replaced by a 40% (w/v) NaOH solution) Reagents must be prepared fresh. Reagents are also re- ferred to as VP-1 and VP-2 or VP-A and VP-B. Sterile culture tubes containing 5 mL of MR-VP broth. Sterile culture test tubes and control bacterial strains (commonly used controls areEscherichia coli and Enterobacter aerogenes) from trypticase soy agar or broth inoculation loop (disposable or have additional equipment available for steril- ization of the inoculation loop) and transfer pipettes. Following method was followed for the test: Add 0.6 mL of Barrett’s reagent A and 0.2 mL of Barrett’s reagent B carefully in a test tube. Shake the tube for 30 s to 1 min, expose the medium to atmospheric oxygen for the oxidation of acetoin to obtain a colour reaction. Allow the tube to stand for at least 30 min. Within 1 h, compare the test result to control cultures to determined culture is VP positive or VP negative. Delayed reading of the result may lead to an erroneous reading, over time a-naphthol and KOH may react to give a copper-like colour. Bacteria fermenting sugars via the butanediol pathway pro- duce acetoin (i.e., acetyl methyl carbinol or 3-hydroxybutanone) as an intermediate which can be further reduced to 2,3 butanediol. In the presence of KOH the inter- mediate acetoin is oxidized to diacetyl, a reaction which is catalyzed by a-naphthol (2). Diacetyl reacts with the guanidine group associated with molecules contributed by peptone in the medium to form a pinkish-red-coloured product. The a-naphthol in
Figure 3.5: Bacterial identification tests in laminar air flow the Barritt’s modification of the VP test serves as a colour intensifier.
8. Methyl red solution:Methyl red and Voges-Proskauer test using MR-VP Medium (Hi- media M070). Following steps were followed: Completely dissolved 0.1 g of methyl red in 300 mL of ethanol (95%). Add 200 mL of deionized water to make 500 mL of a 0.05% (w/v) solution in 60% (v/v) ethanol. Store the prepared methyl red so- lution at 4◦C. Transfer 2.5 mL of culture into a new sterile culture tube. Add 5 drops of the methyl red reagent. Compare the test organism to the control cultures to im- mediately interpret the result. MR positive, false-negative results or an inconclusive orange colour may occur due to insufficient length of incubation. In this case, it is recommended that the test can be repeated with a culture that was incubated for an additional 24 to 48 h. An increase in glucose concentration in the medium might lead to false-positive results in the MR test. Clark and Lubes showed that increased glucose concentration also led to high hydrogen ion concentration in high-ratio cultures.
9. Oxidative-fermentative test used media with glucose: Oxidation-fermentation re- action test using basal medium (Himedia M395), Peptone (tryptone) 2.0 g, Sodium- chloride 5.0 g, Glucose (or other carbohydrate) 10.0 g Bromthymol 0.03 g, Agar 3 g, Dipotassium phosphate 0.30 g. The oxidative-fermentative test is used to determine gram-negative bacteria that metabolize carbohydrates oxidatively by fermentation, or are nonsacchrolytic and therefore have no ability to use the carbohydrate in the me- dia. After the medium is autoclaved at 121◦C for 15 min, a filter sterilized solution of 10% solution of carbohydrate is aseptically added to the medium to a final concentra-
3.3. PHASE II: Microbial Studies on the Best Trial of Water Hyacinth Compost
tion of 1%. The sterile medium containing the carbohydrate is aliquoted aseptically into sterile test tubes and cooled unslanted as stabs. Some procedures call for the addition of 10 g/L of carbohydrate to the medium prior to sterilization. The medium is then dissolved by heating to a boil on a hot plate or by steaming for 20 min prior to aliquoting into test tubes. The medium in test tube is then steamed for 20 min in place of autoclaving to prevent break down of the carbohydrate. Basal medium is commercially available in a premixed form from biological supply companies. The carbohydrate source is not included and must be added as stated above.