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Microbiology Lab Final
Terms in this set (18)
- What are we testing for in the SIM stabs- products produced, what is being broken down?
Testing for hydrogen sulfide production, indole production, & motility. Tryptophan is broken down
What reagents need to be added to the SIM stabs and what are they reacting with; what colors do things turn and what do they mean?
Reagents: Kovac's, 10 drops. Reacting with indole to produce red ring at top of meniscus. Positive is red ring, negative is gold/yellow ring.
Black color means positive for hydrogen sulfide production. (The actual black stuff is iron sulfide, NOT hydrogen sulfide)
- What enzyme is important for starch hydrolysis? What reagent is added to read the plates and what is it reacting with? How do you read the results of the starch plates?
Enzyme important for starch hydrolysis is amylase. Reagent added is Gram's iodine, reacts with starch. Read the results by looking for a "halo" around the bacterial colony. Halo means positive for starch hydrolysis.
· Substrate in IMViCs
Glucose-peptone & sodium citrate
· Any important enzymes mentioned and what they are responsible for doing
Indole: Tryptophan enzyme, produces indole, pyruvic acid, and ammonia.
· Products produced in IMViCs
I: Indole, pyruvic acid, ammonia
M: Acids (formic, succinic, acetic, lactic, pH lower than 7)
Vi: Neutral/basic products (acetone) (acetoin is intermediate product before butyl glycol is produced)
C: Sodium carbonate
How to read the results of IMViCs; including reagents added
I: positive means reacted with indole to produce red. negative yellow/gold (Kovac's reagent)
M: positive red, acids produced (Methyl Red reagent)
V: positive red, base/neutral produced (KOH & alpha-naphthol reagent)
C: positive blue & growth, negative green (bromothymol blue reagent)
· Substrates in the carbs
Sucrose, Lactose, Glucose, Mannitol
· What is a durham tube
Small tube inside of a larger tube that is used to catch gas during fermentation of acids
· Any indicators present
· How to read the results of carbohydrate fermentation
Yellow tube means positive for carbohydrate fermentation. Red tube means negative for carbohydrate fermentation. Gas in durham tube means gas produced.
· Important enzymes mentioned and the reactions they are responsible for (catalase, oxidase, nitrate)
Catalase: superoxide dismutase, neutralizes super-oxide free radicals. Peroxidase, breaks down hydrogen peroxide & does not produce oxygen. Catalase, breaks down hydrogen peroxide into water and oxygen gas.
Oxidase: Cytochrome oxidase, adds spare electrons to oxygen (reduces) & creates water using spare hydrogen atoms in cell.
Nitrate reduction: nitrate reductase, reduces nitrate to nitrite.
· How to read the results of these tests
Catalase: Positive, bubbles form
Oxidase: Positive, yellow
Nitrate: Positive, red. Negative, no change. If negative after 15 minutes, add zinc to test for denitrification.
Denitrification: Positive: No color change. Negative: Red (means nitrate reduction)
How to do dilutions
Original sample: pipette 1 mL into first blank.
First blank (10^-2): Pipette 1 mL into second blank
Second blank (10^-4): Pipette 1 mL into third blank, so on and so forth
· How to read an MPN and the media used
a.) Find series of tubes that give 5-5-5-5-5
b.) Take row next to the last of the positive rows and then take the next row down in the series; you need 3 rows (this becomes your MPN number) (red is positive, yellow is negative)
c.) Using the chart and MPN, determine the number of cells present
d.) Divide number of cells by the dilution factor (DF) (DF= middle dilution of three tubes)
e.) Divide cells by 100 mL (chart is based off of 100 mL)
Example: 5-4-3 = 280
Media used in MPN
How to read a pour plate
Count CFUs (colony forming unit), must be between 25 and 250.
Determine dilution factor (if you counted a plate diluted to 10^-7, then DF is 10^-7)
Example: 135 CFUs in a 10^-7 plate (diluted with 0.1 mL) would be 1.35 x 10^9/mL (decimal is moved in 135 to 1.35 for scientific notation)
· Kirby Bauer Method; media used and technique for reading results
Media used: Mueller Hinton agar
Technique for reading results: Measure zone of inhibition in millimeters and compare to antibiotic chart
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