(Lab 2) Biochemical Tests Flashcards
What biochemical test is shown?
Mannitol Salt Agar (MSA)
What is the purpose of MSA?
- detect if an organism is halotolerant
- detect if an organism can ferment mannitol
What are the ingredients in MSA?
Selective ingredients: 7.5% salt concentration (inhibits non-halotolerant organisms)
Differential ingredients: mannitol (for fermentation)
Does MSA use a reagent or a pH indicator? Describe it
Phenol Red pH indicator:
- yellow (acidic)
- red/orange (neutral)
- pink (alkaline)
What are the possible results in a MSA plate?
No growth: non-halotolerant
Growth: halotolerant
yellow growth: ferment mannitol
pink growth: use of proteins instead of mannitol
What biochemical test is shown?
Eosin Methylene Blue Agar (EMB)
What is the purpose of an EMB plate?
- Selects for G- species
- differentiates organisms that can ferment lactose
What ingredients are in an EMB plate?
Selective ingredients: Eosin and Methylene blue (inhibit G+ growth)
Differential ingredients: Lactose [differentiate coliforms (G-lactose fermenters) from non-coliforms]
Eosin (pH indicator. combines with color of methylene blue to produce results)
Does EMB use a reagent or pH indicator? Describe it.
pH indicator: Eosin:
- metallic green: very acidic (coliforms)
- dark purple: acidic
- pink: neutral/alkaline
What are the possible results in an EMB plate?
What biochemical test is shown?
Phenylethanol Agar (PEA)
What is the purpose of PEA?
Selects for G+ bacteria
What ingredients are in a PEA plate?
selective ingredients: phenylethanol
Does PEA use a pH indicator or a reagent? Describe it.
Neither. Indication is based on growth
What are the possible results for a PEA test?
Strong growth: Probably G+
Weak growth: Probably G-
What bacteria is PEA good for identifying?
G+ Staphylococcus and Streptococcus
What biochemical test is pictured?
Fluid Thioglycolate Broth
What is the purpose of FTB?
Determine the specific O2 requirements of different bacterial species
What ingredients are in FTB? Describe them.
Thioglycolate: bind O2
Resazurin: O2 indicator
- pink: O2 present
- clear - O2 absent
Gelatin: slows diffusion of O2 into media
What are obligate aerobes? What is their appearance in Thioglycollate broth? What enzymes do they have against oxidative damage?
- Use aerobic respiration only
- contain both Superoxide dismutase and catalase
What are facultative anaerobes? What is their appearance in Thioglycolate broth? What enzymes do they have against oxidative damage? Provide an example.
- use aerobic respiration if O2 available and anaerobic pathways if not
- contain both Superoxide dismutase and catalase
- majority of bacteria
What are aerotolerant anaerobes? What is their appearance in Thioglycollate broth? What enzymes do they have against oxidative damage?
- do not use O2 but can survive if present
- contains only Superoxide dismutase
What are microaerophiles? How do they appear in Thioglycolate broth? What enzymes do they have against oxidative damage? Provide an example.
- use aerobic respiration, but require low O2 concentration, ~ 1%
- contains both Superoxide dismutase and catalase at low concentration
- Neisseria gonorrhoeae
What are obligate anaerobes? What is their appearance in Thioglycolate broth? What enzymes do they have against oxidative damage? Provide an example.
- do not use aerobic respiration. O2 is toxic
- contains no enzyme against oxidative stress
- Clostridium difficil
What biochemical test is pictured?
Starch agar (non-selective)
What is the purpose of a starch agar plate?
Determine if a bacteria has the enzyme amylase to digest carbohydrates
Does starch agar use a pH indicator or a reagent?
Reagent: Iodine
What are the possible results with a starch agar?
Black: Iodine + starch
clear: iodine + glucose
What biochemical test is pictured?
Skim milk agar
What is the purpose of the skim milk agar?
Determine if an organism contains contains casease to digest casein protein into amino acids
What are the possible results for skim milk agar?
Clear halo: has casease
no halo: no casease
What biochemical test is pictured?
gelatin deep
What is the purpose of a gelatin deep?
determine if an organism produces the exoenzyme gelatinase
What are the possible results for gelatin deep?
Solid: negative
liquid: positive
What biochemical test is pictured?
Oxidase test
What is the purpose of the oxidase test?
detect a specific cytochrome oxidase found in the electron transport chain of some bacteria
What is significant info for Enterobacteriaceae and oxidase test?
They are all oxidase negative
Does the oxidase test use a reagent or a pH indicator? Describe it
Reagent: Oxidase reagent by cytochrome oxidase
What precautions are taken to prevent a false positive in the oxidase test?
- do not use metal
- look at results within 30 seconds
- do not pick up agar
What are the possible results you could see for the oxidase test?
Positive: purple/blue
negative: no color
What biochemical test is pictured?
catalase test
What is the purpose of the catalase test?
Looking for the presence of catalase in certain bacteria
What category of bacteria contain catalase?
Most aerobic and facultative anaerobic bacteria
What are the possible results you can see in the catalase test?
gas bubbles: positive
no bubbles: negative
What is the catalase test able to distinguish best?
Distinguish between hemolytic G+ Enterococcus (neg), Streptococcus (neg), and Staphylococcus (pos)
What biochemical test is pictured?
Carbohydrate fermentation test
What is the purpose of a carbohydrate fermentation test?
determine if a microbe can ferment a specific sugar
What are the ingredients are in the carbohydrate fermentation test?
- 1 sugar (glucose, lactose, or mannitol)
- Phenol red pH indicator
- durham tube for gas collection
Does the carbohydrate fermentation tube use a pH indicator or reagent?
pH indicator: Phenol red
- yellow: pH~6
- red/orange: pH~7
- pink: pH~8
What are the possible results you can see with the carbohydrate fermentation tube?
Yellow: positive (+A/+AG)
- sugar fermentation with or without gas
Orange/red: negative (-)
- no sugar fermentation
pink: negative (-alk)
- no sugar fermentation
- protein digestion
What biochemical test is pictured? What is its purpose?
IMViC: Indole test
- to determine whether bacteria can break down tryptophan. Produces indole as a by-product
Does indole use a pH indicator or a reagent?
No pH because ammonia and pyruvate neutralize.
Reagent: Kovac’s reagent
- Kovac’s reagent=yellow
- Kovac’s reagent + indole=red
What are the possible results with indole?
Red: positive
Yellow: negative
Why is IMViC an important ID procedure after Gram stain?
Differentiates between members of Enterobacteriaceae
What biochemical test is pictured? What is its purpose?
Methyl Red, Vogues-Proskauer (MRVP)
determine whether bacteria ferment glucose into mixed acids (MR) or acetoin (VP) (mutually exclusive)
Does MR use a pH indicator or a reagent?
pH indicator: Methyl red
What results can you expect with an MR test?
Red (pH ≤ 4) - MR+
No color (pH > 4) - MR-
Does VP use a pH indicator or a reagent?
Reagent: Barritt’s reagent A & B
What results can you expect with a VP test?
Reagents alone produce copper color
Reagents turn red in the presence of acetoin
How long does MRVP broth need to incubate? Why?
48 hours because acetoin is produced within that time for non-acidic fermentation. Less than that, it can give a false negative since acids will be produced first
What biochemical test is pictured? What is its purpose?
Citrate test: Determine if bacteria can use citrate as a sole carbon source. Organisms won’t grow if they can’t use citrate.
If organism grows it uses protein in media to produce alkaline substances
Does the citrate test use a pH indicator or a reagent?
pH indicator: Bromothymol Blue
What results can you expect with a citrate test?
Blue tube (alkaline pH) OR any growth: positive
Green tube and no growth (neutral pH): negative
What biochemical test is pictured? What is its purpose?
Hydrogen sulfide test - test if an organism can produce H2S from cysteine (sulfur-containing amino acid)
Cysteine desulfurase
What media is used for the H2S test? What are its ingredients?
Peptone iron deep
- peptone: Source of cysteine
- iron: Binds to H2S to visualize results
Why is a deep used for H2S test?
To simulate anaerobic conditions
Does H2S test use a reagent or a pH indicator? Why?
Reagent: iron
Pyruvate (acidic) and NH3 (basic) produced from the breakdown of cysteine neutralize each other
What biochemical test is pictured? What is its purpose?
Urease test: determine if bacteria can hydrolyze urea (urease)
What media is used for Urease test? What are the ingredients?
Urea slant
- urea as substrate
- phenol red as a pH indicator
Does the urease test use a pH indicator or reagent? Why?
pH indicator: phenol red
CO2 acidic and NH3 basic
What results can we expect with the urease test?
yellow-acidic: negative
red/orange-neutral: negative
pink-alkaline: Positive
How might urease activity be a virulence factor?
Urease activity for H. pylori neutralizes stomach acids which can cause ulcers. In P. vulgaris, it allows the bacteria to grow in the presence of urine and can cause diaper rash NH3 burn
What is the purpose of nitrate reduction test?
determine if bacteria can reduce nitrate.
Does nitrate reduction use pH indicator or reagent?
Reagent A+B and Zinc
What possible results can you have with nitrate reduction test?
NO3- present: negative (red)
NO2- present: pos (red)
N2 or NH3 present: pos (yellow)
What biochemical test is shown? What is its purpose?
Lysine Decarboxylase test: determine if bacteria can decarboxylase lysine
What is the media used for Lysine Decarboxylase test? What are the ingredients?
Lysine broth
- Lysine -> alkaline products
- glucose -> acidic products
- Bromcresol purple: pH indicator
What are the expected results of Lysine Decarboxylase test?
What biochemical test is shown? What is its purpose?
Phenylalanine deaminase test: determine if bacteria can deaminate phenylalanine
Does Phenylalanine deaminase test use a pH indicator or reagent?
reagent: Ferric Chloride (FeCl3)
What are the expected results of Phenylalanine deaminase test?
Which tests can be used to differentiate species of Enterobacteriaceae?
Lactose fermentation
IMViC
Hydrogen Sulfide
