Micro Lab Practical 2
What media is used for the citrate utilization test?
Simmons citrate agar slant
- Bromothymol blue - pH indicator
- Citrate - source of carbon
- Ammonium ion (NH4) - Nitrogen source
What is the citrate utilization test used for?
To determine if an organism can use citrate as the sole source of carbon
What are the by-products of citrate utilization?
NH4 -> NH3 (elevated pH)
What are the observations and results of Citrate utilization?
Blue color - Positive result
Green Color - Negative result
What is the interpretation of Citrate utilization?
Positive = produces citrate permeate and uses citrate as the sole carbon source
negative = Does not produce citrate permeate and cannot use citrate as the sole carbon source
What is the media used in glucose fermentation?
Broth agar
Glucose - carbohydrate
Phenol red - pH indicator
Peptone - protein (amino acid) digestion
Durham tube
What is the Glucose fermentation test used for?
To determine if microbes are capable of using fermentation to metabolize glucose (carbohydrates)
What are the by-products of Glucose Fermentation?
CO2
Ammonia (elevated pH)
What are the observations and results of Glucose Fermentation?
Yellow = positive
red = negative
Pink in tube = positive for deamination
Yellow in tube = positive
What is the interpretation of Glucose Fermentation?
Yellow = ferments sugar
Red = negative for fermentation
Pink inside/outside the tube = positive for deamination and digests proteins
Yellow inside tube = quick fermenter and ferments sugar
What is the media used for nitrate Reduction?
- Beef extract, peptones, nitrates, and other nutrients
- Durham tube
What is the purpose of the Nitrate Reduction test?
To test if the microbe is capable of reducing nitrate
What are the byproducts of Nitrate reduction?
Nitrite (NO2-)
Nitric Oxide (NO)
Nitrous Oxide (N2O)
Nitrogen Gas (N2)
What are the observations and results of Nitrate Reduction?
Bubbles in Durham tube -> positive
No bubbles: adding reagents A and B make it red -> Positive
No bubbles: adding reagents A and B make no change: No color change with Zinc -> positive
What is the interpretation of Nitrate Reduction?
Bubbles in Durham tube -> positive for denitrification
No bubbles: adding reagents A and B make it red -> Positive for nitrite reduction; partial reduction
No bubbles: adding reagents A and B make no change: No color change with Zinc -> positive for either nitric oxide or nitrous oxide; partial reduction
What is the media used in Urea Utilization?
Urea agar (peach colored)
Urea
Phenol red
Glucose
What is Urea utilization used to test?
If the bacterium can produce the enzyme urease and hydrolyze urea
What is the byproduct of the urea utilization?
Deamination
Urea -> NH3 (ammonia)
Elevated pH
What are the observations and results of Urea Utilization?
Hot pink -> positive (Basic)
Peach/yellow -> negative (Acidic)
What is the interpretation of urea utilization?
Positive = hydrolyzes urea and is positive for the production of urease
Negative = Does not hydrolyze urea and is negative for the production of urease
What is the media used in Mannitol Salt Agar?
Mannitol Salt Agar
7.5% salt
Phenol red
Mannitol - carbohydrate (sugar)
What is the purpose of Mannitol Salt Agar?
To test for the presence of halophiles and the fermentation of mannitol
What is the byproduct of Mannitol Salt Agar?
elevated pH
What are the observations and results of Mannitol Salt Agar?
Growth + yellow color = positive
Growth + red color = positive and negative
No growth = negative
What is interpretation for Mannitol Salt Agar?
Growth + yellow color = halophile, mannitol fermenter
Growth + red color = Halophile, not a mannitol fermenter
No growth = not a halophile
What are the 3 microbes used for the Mannitol Salt agar and what are their results?
Escherichia coli - No growth - not a halophile
Staphylococcus Saprophyticus - halophile: ferments mannitol
Staphylococcus epidermidis - halophile: does not ferment mannitol
What is the media used for Beta-Lactamase?
Nitrocefin disc
What is the purpose of the Beta-Lactamase test?
To determine if the organism is susceptible or resistant to B-Lactam drugs
What is the byproduct of the Beta-Lactamase test?
NONE
What are the observations and results of the Beta-Lactamase test?
Pink Disc - Positive
No color change - Negative
What is the interpretation of the Beta-Lactamase test?
Pink Disc - Produces B-Lactamase and is resistant to B-Lactam drugs
No color change - Does not produce B-Lactamase and is not resistance to B-Lactam drugs
What is the media used in the Kirby-Bauer Test?
Mueller Hinton Agar
What is the purpose of the Kirby-Bauer Test?
To test a bacterium's susceptibility to a specific antibiotic
What is the byproduct of the Kirby-Bauer Test?
Nothing
What is the Observation and interpretation of the Kirby Bauer test?
The zone of inhibition - area of no bacteria growth
If zone is more than the stated threshold = susceptible to the antibiotic
If the zone is less than the state threshold = resistant to the antibiotic
What is the media used in the Use dilution test?
Agar broth
What is the purpose of the Use Dilution test?
To determine the MIC of a disinfectant or antiseptic that will effectively inhibit the growth of the bacteria
What are the observations and results of the Use Dilution test?
Positive control - tests for viability
Negative control - tests for sterility of bead
Concentration is clear - lowest clear concentration is the MIC
What does the B-Lactam drug do?
Interfers with the peptidoglycan synthesis in growing and reproducing bacterial cells
What happens if a microbe contains the enzyme B-Lactamase?
They can render B-Lactam drugs ineffective
How are the Mueller Hinton agar plates poured for the Kirby-Bauer tests?
With an exact amount of agar so every plate is identical and poured to the same depth to assure equal diffusion
What is the difference between antiseptics and disinfectants? which test tests for this?
Antiseptic - chemicals that inhibit microbial growth on living tissue
Disinfectant - Chemicals that inhibit microbial growth on surfaces
The MIC of the solutions are at a concentration that is ____ and ____ to handle
Effective and safe
MSA is selective for _____
MSA is differential for _____ _____
Halophiles
Mannitol fermentation
What are resident microbes?
Gram-positive bacteria and yeasts that are harmless under normal conditions
What are transient microbes?
Easily removed from the skin, and are highly varied (gram+ to Gram -)
What is the enzyme Citrate permease do?
Transport citrate into the cell and metabolize it
What happens when we add reagents A and B to the Nitrate Reduction test and it turns red?
It has the enzyme nitrate reductase and has reduced nitrate to nitrite
Why is zinc to pink and negative result for nitrate reduction?
Zinc is a catalyst and it will reduce any nitrate present to nitrite. The zinc caused the reaction. If the organism was able to reduce nitrate, there wouldn't be any in the tube
what is a selective medium?
specialized medium that contains the ingredients that inhibit the growth of some microbes and encourage the growth of others
What is a differential medium?
specialized medium that contains ingredients that allow microbiologists to distinguish between different species of bacteria by visual observations
What are biochemical tests?
Used to identify species of microbes by differentiating them on the basis of their biochemical activities