This test uses a differential stain called gram stain. The first step of this test is to apply crustal violet stain to the slide containing the heat fixed bacteria. Next, iodine is added to further enhance the crystal violet stain by forming a crystal violet-iodine complex. The most critical step in the test is decolorization by washing the slide in alcohol. The resulting color or lack of color determines whether the bacteria is Gram positive or gram negative. Gram positive bacteria are not decolorized by the alcohol due to the alcohol which makes their cell wall less porous and retains the crystal-violet stain. On the other hand, Gram negative bacteria are decolorized by the alcohol because their cell walls become more porous and cannot retain the crystal violet stain. A counter stain, safranin, is then used to differentiate the gram positive and gram negative cells. Gram positive bacteria stain purple while Gram negative bacteria stain red.

This test involves taking the slide with the Gram positive or Gram negative bacteria and applying immersion oil. The oil increases the resolution of the bacteria while looking through the low power magnification lens. You then observe the shape of the bacteria, rod or cocci. The cell shape leads you to the next test of the flow chart in the Micro. Lab manual.

This test identifies motility in bacteria. By using the stab inoculation technique the bacteria is transferred into the motility agar tube. The agar has low concentration of nutrients in comparison to nutrient rich agar. Motile bacteria grows outwards from the stab towards the edges of the test-tube.

This test examines whether the bacteria cells can retain the primary stain after being treated with acid-alcohol. Acid-fast bacteria cells are saturated with thick waxy material called mycolic acid. This make smearing the bacteria to a glass slide difficult. More bacteria is needed to be mixed with a drop of water. The smear is treated with Carbolfuchsin as the primary stain. It is lipid soluble and penetrates the cell wall. Steam heating further enhance the stain by ‘driving’ it into the cell wall. The slide is then washed in alcohol which decolorizes non acid-fast cells. The acid-fast cells retain the stain. A counter stain, methylene blue, is then applied which turns acid-fast cells reddish-purple and non acid-fast cells blue.

This tests uses a differential stain which indicates the presence or lack of spores in bacteria cells. Because spores are made to survive in lean environments, extreme measures must be taken to ensure that the stain can be soaked into a spore. They are largely chemical and heat resistant. Malachite green is washed over a slide containing a particular bacteria. The stain is fixed by steaming procedures. Due to Malachite’s solubility in water and lack of affinity towards cellular material. A counter stain, safranin, is used to stain for example, vegetative cells. The slide’s bacteria is covered in oil immersion and examined under low power in the microscope. Spores are looked for in the cells. Green dots mean the bacteria is endospore positive.

This test proves whether a bacteria cell can produce catalase. Many bacteria produce hydrogen peroxide due to the transfer of electrons from flavoprotein to oxygen. Catalase is used by these bacteria to break down hydrogen peroxide into water and oxygen. This is important for bacteria because hydrogen peroxide damages cell components and the ability to produce catalase allows the bacteria to live in a more ranged environments. By adding hydrogen peroxide to a test-tube containing agar and an unknown isolate the resulting reaction of bubbles proves the bacteria be catalase producing positive. No bubbles indicates a negative catalase producing bacteria.

This test is performed by stab inoculating a TSA slant with an unknown isolate. You incubate and then observe in two days. Bacteria growth on only the top of the slant indicates aerobic bacteria. Facultative aerobic bacteria grow on top and further down the stab. Bacteria growth on only the bottom of the slant indicates an anaerobic bacteria.

The test uses Simmon citrate agar which is a medium that only allows sodium citrate as the sole carbon source and ammonium ion as the sole nitrogen source. A slant is used because the test is an aerobic process and the surface is required to be exposed to air. A chemical BTB is also included in the agar as a ph indicator. Citrate is used by some bacteria as a carbon source if fermentable carbohydrate is not present. Citrate permease transports the citrate into the cell which is thin broken down. Carbon dioxide is a waste that is produced from breaking down citrate, this reacts with the sodium and water in the Simmon citrate agar to produce sodium carbonate. This causes the alkaline to be produced which increases the production of acid that affects the BTB and turns the agar a deep blue.

The MR test is performed to examine whether a bacteria can ferment glucose into stable end acid products. The VP test identifies bacteria capable of 2,3-butanediol fermentation. Both tests involve using mixed-acid reactions. Bacteria is added to the MR broth and incubated. If the broth turns red it indicates a bacteria that can produce enough stable acid products which overcome the buffers in the broth and lower the ph. Yellow equals a negative test result. On the other hand some bacteria produce less acidic end products for example, 2,3-butanediol. Barrit reagents are added to the broth to react with the precursor to 2,3-butendiol, acetoin. Red indicates a positive test while yellow indicates a negative test.

This test indicates the ability of a bacteria to hydrolyze urea. Urease hydrolyzes urea so the resulting ammonia can be used as a nitrogen source. Bacteria is added to a urea medium that is liquid. The medium contains urea and phenol read a ph indicator. Ammonia increases, raises the ph. A pink color indicates that the ph is above 8.4 and turns the medium pink, indicating a positive test result. Medium remaining pink indicates a negative test result.

This test indicates the presence or lack of production of tryptophanase. A bacteria that can hydrolyze tryptophan results in the production of indole, pyruvic acid, and ammonia. Bacteria is added to a liquid medium of 1%tryptone and incubated. Kovac’s reagent is added to the medium. During the hydrolysis of tryptone, indole is produced which forms of red layer of para-dimethylaminobenzaldehyde that floats on the liquid mediums surface. This red layer indicates a positive indole test. No layer indicates a negative result that means the bacteria is unable to produce the enzyme tryptophanase.

Phospholipid cell membranes contain lipids. These lipids and other materials are broken down to produce energy for the cell. Hydrolytic liapases hydrolyze the lipids. By breaking down large lipids they are able to pass through the cell and become a supply of carbon and energy. Bacteria is added to a egg yolk agar plate and allowed to incubate for two days. If an opaque ring forms around the area that the bacteria was inoculated to then the bacteria is able to hydrolyze lipids. If no ring then the bacteria cannot use the energy stored in lipids.

This tests to indicate whether a bacteria has amylases which are enzymes that breakdown a polysaccharide starch into a monosaccharide glucose. Starch is prevalent in many environments but it is too large for many bacteria to allow it to pass through their cell membrane. Amylases accomplish the task of breaking the starch into mono and disaccaharide sugars. These are then used for energy. Bacteria is inoculated onto a agar plate and allowed to incubate. Iodine is then flooded onto the plate. Deep purple to brown indicates iodine is reacting with starch. Clear areas around the bacteria indicate the use of amylases, positive test result. Absence of clear area is a negative test that mean the bacteria was unable to hydrolyze starch.

This test indicates whether a bacteria can reduce nitrate to nitrite. Two systems reduce nitrate for the use as an final electron acceptor of the cytochrome system which produces a reduced nitrogen compound. Anaerobic respiration and dentrification are the systems that reduce nitrate. Bacteria is inoculated into a nitrate broth that contains some agar to slow oxygen diffusion and encourage anaerobic growth while incubating. Barrit’s nitrate reagent are added to the solution to detect nitrite presence. It reacts with the nitrite that then produces nitrous acid which reacts with the reagent. This reaction forms a red color as an indicator of positive nitrate reduction. No color change means that the organism cannot reduce nitrate.

This test indicates whether a bacteria can catabolize carbohydrates. This process is important because carbohydrates are great potential sources of energy. Bacteria is inoculated into phenol red broths that contained small gall tubes inside the test-tube. Each broth has either dextrose, sucrose or lactose as the sole fermentable nutrient. As the bacteria ferment they produce gas and acid. These two indicators distinguish bacteria. If acid is produced then the broths turned from red to yellow to indicate a positive acid result. If gas was produced during fermentation air would appear inside glass tubes submerged in broth in larger test-tubes. Each particular test-tube had a different fermentable carbohydrate which differentiated which bacteria could ferment certain carbohydrates.

This tests indicates whether a bacteria can produce hydrolytic enzymes called gelatinases to digest and liquify gelatin. Gelatin is a protein potential energy source which is to large for most cells to allow into their semi-permeable membranes. As a result these bacteria produce gelatinanses to breakdown the gelatin and use it for energy. A gelatin test-tube is stab inoculated with a needle carrying bacteria. A positive result is for the bacteria to liquify the gelatin and a negative result is for the gelatin to remain solid. The test-tube with gelatin must be kept at 28 degrees or else the gelatin will melt and liquify with no input from bacteria.