Microlab ecu practical
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PEA agar (Phenyl Ethyl Alcohol Agar
- • Selective for Gram + bacteria, inhibits the growth of Gram – bacteria
- • Phenyl ethyl alcohol inhibits DNA synthesis of G – organisms, which is why they fail to grow
MSA agar – Mannitol Salt Agar
• Contains a high concentration of salt which inhibits the growth of Gram – bacteria and most Gram + bacteria; selective for halophilic bacteria
Pseudomonas P Agar
- • Selective for Pseudomonas spp. Other Gram- and Gram + bacteria will not usually grow on this agar.
- • If the Pseudomonas spp that grow on the plate produce pigment, the agar color will change from a dull beige color to anywhere from a light green to a dark blue. This is a positive reaction for pigment production for Pseudomonas aeruginosa, our test strain.
- • Selective for Gram – bacteria as the dye crystal violet and bile salts inhibit the growth of Gram + bacteria
- • Differential in that it contains the carbohydrate (sugar) lactose and a pH indicator. If lactose is fermented, colonies will appear pink (+ reaction).
- • Tests for the presence of the exoenzyme amylase.
- • Amylase breaks down starch into smaller compounds that the bacteria can import into the cell and use. Starch media is nutrient agar supplemented with exogenous starch.
- • When we pour Iodine on the plate after the bacteria have grown, the iodine reacts with the starch present in the media, turning it black.
- • If the bacterium is amylase + and can break down starch, a clear area will appear around the region where the bacteria grew. The clear zone indicates the absence of starch and the presence of the amylase enzyme.
Spirit Blue agar
- • Tests for the presence of lipase, an exoenzyme that breaks down lipids
- • + test indicated by a zone of clearing surrounding the bacterial growth
- o Spirit blue dye in the medium will tend to migrate towards areas of lipid clearing; is considered an indicator of lipolysis
- However, the dye is heat labile and breaks down after approximately 36 hours in the incubator, so the migration of the dye cannot be considered a definitive positive test. It is best to hold the plate up to the light and compare to an un-inoculated plate.
- what else???
OF Glucose Medium
• Determines whether an organism is oxidative (O) or fermentative (F) or both (OF), which means it determines if the sugar can be fermented in the presence or the absence of oxygen.
- • If both tubes were still green, the test is negative, as no fermentation of glucose occurred.
- • If the non-mineral oil tube (the – tube) was yellow, but the mineral oil tube (the + tube) remained green, the organism was considered oxidative (O), meaning it could only break down the sugar in the presence of oxygen.
- • If the +mineral oil tube was yellow, but the non-mineral oil tube remained green, the organism was considered fermentative (F), meaning it could only break down the sugar in the absence of oxygen.
- • If both tubes were yellow, the organism was considered to be oxidative/fermentative (OF), meaning it could ferment the sugar in both the presence and absence of oxygen
Carbohydrate Fermentation Tubes
- • contains a carbohydrate (in lab we tested sucrose and lactose), a pH indicator, and a Durham tube (little upside down test tube within the big test tube)
- If fermentation occurs, it will result in a change from a red color to a yellow color. We record this as “A” for a positive reaction for acid production.
- • If gas is produced during fermentation, a gas bubble will become trapped in the Durham tube. We record this as “G” for a positive reaction for gas production.
- • Bacteria that cannot ferment may grow in the media, but there will be no color change,
- o The MR test measures the end products of the fermentation reaction. If it stays red, acid end products were produced. If it turns yellow, neutral end products were produced
- o The maintenance of a red color is considered a + test, and the disappearance of a red color is considered a negative result.
- ---A + test is indicated by the formation of a reddish ring at the broth oxygen interface at the top of the tube.
Nitrate Reduction Test
- • Tests ability of organism to reduce nitrate (NO3) to nitrite (NO2) or further reduction to ammonia (NH3) or nitric oxide (NO)
- • After incubation, add Nitrate A and B reagents. The formation of a red color throughout the broth tube is a positive test
- o A positive test indicates NO3 (nitrate) was reduced to NO2 (nitrite). (Nitrate reagents A and B specifically detect nitrite (NO2).
- • Therefore, if no color change was observed in the first reaction, we must add zinc dust to confirm one of the two above possibilities
- o Zinc reacts with NO3 (nitrate) and causes a red color change. If this happens, no reduction has occurred since NO3 is still present in the tube, and the reaction is considered negative.
- o If no color change occurred after adding the zinc dust, then the organism did reduce, but reduction was not to nitrite but to some other nitrogen containing compound, and the reaction is considered positive.
- • If no color change occurred after the addition of Nitrate A and B reagents, two possibilities exist.
- o No reduction occurred and nitrate is still present in the tube.
- o NO3 (nitrate) was reduced even further, i.e. past NO2 (nitrite)
- • Nitrate-positive if nitrite (pink color) is detected
- Nitrate-negative if nitrite is not detected (no color change)
- • Tests for the presence of the enzyme catalase
- • Catalase breaks down H2O2 (hydrogen peroxide) to oxygen and water.
- • If a bacterium is catalase - , no bubbles will form
- • Tests for the presence of cytochrome c,
- • A positive test is indicated by the formation of a blue color after the addition of the oxidase reagent
- • A negative test is indicated by the no color change
Gelatin Hydrolysis test
- • Tests for the presence of the enzyme gelatinase
- • If an organism is gelatinase +, it will liquefy the solid gelatin in the tube
- • If an organism is gelatinase -, the gelatin will remain solid
- --if any portion of the tube appears to “run”, the test is considered a positive.
- the presence of the enzyme urease
- • A positive test is indicated by a color change from a light orange to a hot pink color (fuchsia)
- • Urease breakdown of urea generates NH3 (ammonia) as a by-product, which raises the pH of the broth medium.
- • Urea broth contains phenol red. The pH of prepared urea medium is 6.8, so phenol red is yellowish-orange
- • As ammonia builds up in the medium, the pH goes up, and the phenol red indicator changes color form yellowish orange to hot pink.
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