Lab Exam 2 Flashcards
1
Q
Fluid Thioglycollate Media (FTM)
A
- test oxygen requirements and motility
- O2 is reactive - makes reactive oxygen species
- can easily steal electrons
- autoclave to remove O2 from media. Allow to redissolve down the tube
- resazurin - O2 indicator. should be pink at the top of the tube
- do not shake the tube
2
Q
Levels of oxygen requirements
A
- aerobic - bacteria use O2 as terminal electron acceptor in electron transport chain part of aerobic respiration
- strict aerobe - requires O2
- strict anaerobe - cant grow with O2
- fermentation or alternate e- acceptor
- facultative anaerobe - can live with or without O2
- fermentation or respiration
- aerotolerant - can live with or without O2
- only fermentation
3
Q
FTM tube interpretation
A
facultative anaerobe - throught the whole tube
strict aerobe - top of the tube
strict anaerobe - bottom of the tube
4
Q
Mannitol Salt Agar (MSA)
A
- selective - salt tolerance
- differential - mannitol metabolism
- Plate components: mannitol, NaCl 7.5%, phenol rd
- pH >= 7 red
- pH < 7 yellow
- Staphylococcus is 7.5% salt tolerant
- Staphylococcus aureus can ferment mannitol
- ex. growth - salt tolerant - red color - pH >= 7 - no fermentation of mannitol
- id as Staphylococcus and NOT S. aureus
5
Q
halobacterium salinarium
A
obligate halophile
requires 13% NaCl
could not be isolated using MSA plates
6
Q
Blood Agar Types of Lysis
A
- beta - complete lysis - zone of clearance because they burst
- s. pyogenes
- Alpha - insert into membrane and leak heme. Green appearance. Color comes from oxidation of heme
- Gamma - no pores, no hemolysis
7
Q
Blood Agar Theory
A
- some bacteria produce hemolysins that are toxins to red blood cells
- the bacteria tested are fastidious organisms - difficult to grow, need lots of nutrients
- pore forming toxins cluster and insert into RBC
- Streptolysin O
- Streptolysin S
- Pneumolysin
- must stab the agar in order to introduce bacteria to anaerobic activity. Pore forming toxins are aerobe labile (less active around O2)
- Several species can cause the same type of lysis
8
Q
MacConkey
A
- selective - bile salts and crystal violet
- gram neg persist and gram positive die
- bile salts disrupt the cell membrane
- differential - ability to ferment lactose
- neutral red indicator
- lactose metabolism - byproduct is lactic acid which lowers pH and the indicator will be red below 6.8
- if lactose not metabolism then amino acids metabolized which increases pH and colorless/white
- peptone as amino acid source
- peptone, lactose, bile salts, CV, NaCl, neutral red, agar
9
Q
Sorbitol MacConkey
A
- Use sorbitol as carbohydrate
- certain strain of E Coli produces the shiga toxin (from HGT)
- only this strain cannot ferment sorbitol
- white colonies on the plate
10
Q
SIM
A
- Sulfur Indole Motility
- sulfur and indole are differential components
- casein peptone - protein digest rich in tryptophan
- sodium thiosulfate - can be reduced by sulfar
- ferrous sulfate - iron containing compound
- 0.4% agar - gelling agent
11
Q
SIM theory
A
- sulfur can be the terminal electron acceptor
- SO4 to H2S
- H2S + FeSO4 = FeS + H2SO4
- FeS is black
- tryptophanase breaks down tryptophan and produces indole
- indole reacts with Kovacs reagent and turns red
- All black tube indicates motility and it produces H2S
- If you were to add TTC then red could be misinterpretted as there are 2 sources of red indicator
12
Q
TSI/KIA
A
- Triple Sugar Agar / Kligler’s Iron Agar
- fishtail streak and stab
- differential medium
- can it ferment the CHO - slant and butt color
- can it produce gas during fermentation - cracks in agar
- does it reduce sulfur - black product
- reduction of sulfur can only occur under acidic conditions
- black precipitate - sulfur was reduced and fermentation made an acidic environment
- help to identify Gram neg bacilli
13
Q
KIA more info
A
- glucose 0.1% and lactose 1%
- must exhaust all glucose in order to see if it can ferment glucose
- Beef extracts to provide amino acids for possible deamination
- phenol red pH indicator
- thiosulfate - sulfar can be reduced
- ferrous sulfate - can react with H2S to make FeS (black)
- cysteine - sulfar containing amino acid
- agar - gel agent
14
Q
Reporting KIA
A
- slant/butt then gas then sulfur
- K - alkaline
- A - acid
- +/- G for gas
- +/- H2S for sulfur reduction
- example: K/A +G -H2S
- phenol red
- pH< 7 yellow
- pH = > 7 red
- K/A - ferment glucose which produces acidic byproducts then does not ferment lactose so it deaminates AA and raises pH to give a red slant
- A/A - ferment glucose then lactose - both make acidic by products
- K/K - doesnt ferment glucose or lactose
15
Q
Bile Esculin
A
- selective - bile salts
- differential - esculin. Can the bacterium hydrolize esculin in the presence of bile
- differentiate between Enterococci and Streptococci
- makes esculetin that reacts with Fe3+ to make black prec.
- at least half of the tube must be black
- esculine hydrolysis is more common without the presence of bile
- bile conc is different than in McConkey
16
Q
Phenol Red Carbohydrate Broth
A
- single carbohydrate
- phenol red - pH < 7 yellow
- pH = 7 red
- pH > 7 purple/fuscia
- differential test - based on metabolism of the carbohydrate
- Durham tube - determine if bacterium produces gas during fermentation. Bubble must be at least 10% of the tube
- trypticase (AA), NaCl, beef extract, phenol red, CHO
- AA allows bacteria to survive if cant ferment the CHO
- Reversion - if over incubate then use all sugar and AA deamination increases pH and false negative
- purple/fuscia with gas
- Abbreviations: A, K, or - for no change, then +/- G for gas
17
Q
Catalase Test
A
- 2H2O2 = 2H2O + O2
- H2O2 produced during aerobic respiration
- catalase detoxifies the very reactive H2O2
- Differential test between staphylococcus (positive) and streptococcus (negative)
- add H2O2 - bubbles are positive for catalase
- strict anaerobe will not possess catalase - H2O2 not produced because only anaerobic respiration
18
Q
Oxidase Test
A
- differential test
- test for cytochrome c oxidase - transfers electrons from last protein in chain to O2
- artificial electron acceptor is reduced by cytochrome c oxidase and turns purple
- purple - positive
- yellow - negative
- differentiate between pseudomonas (positive) and enterobacteriaceae (negative)
19
Q
Methyl Red
A
- pH < 4.4 red (positive)
- pH > 6.2 yellow (negative)
- pH 4.4 - 6.2 orange
- testing for very stable acids that are byproducts of mixed acid fermentation
- differentiate enteric Gram - bacteria
20
Q
Voges Proskauer
A
- Butanediol fermentation produces acetoin and 2,3 butanediol
- unstable acids that are quickly neutralized
- Add NaOH and alpha napthol as reagants
- red is positive
- copper/yellow is negative
21
Q
pH indicator
A
22
Q
Citrate Test
A
- defined medium
- utilization medium - essential nutrients are limited
- citrate as sole C source
- ammonium phosphate as sole N source
- Bromthymol blue
- pH 6.9 green
- pH 7.6 blue
- organisms that can use citrate and ammonium phosphate will produce ammonia and raise the pH - blue indicator
23
Q
IMViC
A
Indole
Methyl Red
Voges Proskauer
Citrate Test
