Micro Basics (media, microscopy, etc)

Question 1

AST results are dependent on:

Answer 1

1) Inoculum density
–standard density should be used
2) pH of media
3) Cation concentration of media
4) Supplements / abx in media
–NaCl
–lysed blood
5) Thymidine content in media
6) Incubation conditions
–CO2, O2, temp
7) Incubation length
8) Concentration of abx

Question 2

Media cation concentration that is too high results in __ zone sizes

Answer 2

Smaller

Question 3

Cefoxitin is a surrogate for __ in S. aureus testing?

Answer 3

mecA mediated oxicillin R

Question 4

Media that is too thin results in __ zone sizes

Answer 4

too large

Question 5

Antibiotic concentration gets smaller/larger as distance from the disk increases

Answer 5

Smaller. The highest concentration is next to the disk (abx diffuses out).

Question 6

What is the most common reason for antibiotic conditions to fail?

Answer 6

1) Bag/jar not sealed
2) Catalyst inactive

Question 7

SPS is used for blood culture collection because…

Answer 7

1) anticoagulase
2) inactivates neutrophils
3) inactivates some abx
4) precipitates some complement

Question 8

Waiting more than 15 minutes to incubate the plate after dropping abx disks leads to larger / smaller zone sizes?

Answer 8

Larger

Question 9

Campy need what gas concentrations to grow?

Answer 9

5% O2, 10% CO2, 85% Nitrogen

Question 10

Bacteria with capsules

Answer 10

Some Killers Have Pretty Nice Capsules Yo Bebe
Strep pneumo
Kleb
H flu b
Pseudo aeruginosa
Neisseria men
Crypto neoformans / Cholera
Yersinia pestis
Bacillus anthracis
Bordetella pertussis

Question 11

Bacteria with cAMP toxins

Answer 11

CAMP
Cholera
Anthrax
E coli (M turned sideways)
Pertussis

Question 12

Bacteria with phage vir factors

Question 13

Urease pos orgs

Answer 13

PUNCH KISS
Proteus
Ureaplasma
Nocardia
Cryptococcus
Helicobacter
Klebsiella
Staph saprophyticus
Staph epidermidis

Question 14

Oxidase pos orgs

Answer 14

VP CHEN LAMB
V. cholera
Pasteurella / Pseudomonas / Plesiomonas
Campylobacter
H. pylori
Exclude Enterobacteriaceae
Neisseria

Legionella
Aeromonas
Moraxella
Brucella

Eikenella
Kingella

Question 15

IMViC

Answer 15

Indole/Methyl Red/Vogues Proskauer/Citrate
1) Indole
–Peptone broth + Kovac’s reagent = red on top of tube
2) MR
–5.0 ml buffered peptone broth
–Acid from glucose fermentation
–48 hour incubation then Methyl Red indicator = Red+
3) VP
–Peptone broth from MR test (before MR added)
–Mixed acid fermentation from glucse
–48 hour incubation then alpha-naphthol + KOH = Red on top+
4) Citrate
–Bromothymol blue + Simmons agar
–Blue after incubation = +

Indole+ = Ecoli / P. vulgaris / K. oxytoca
MR= = Kleb / Enterobacter
MR+/VP+ = S. aureus
VP+= Kleb/Enterobacter/Moraxella
Cit+ = Salmonella / Kleb / P. mirabilis / Citrobacter / Enterobacter / Serratia

Question 16

Citrate positive

Answer 16

VPPPECKSS
Vibrio cholera
P. mirabilis / Providencia / Pseudomonas
Enterobacter
Citrobacter
Kleb
Salmonella / Serratia

Question 17

Citrate negative

Answer 17

SEEY
Shigella
Ecoli
Edwardsiella
Yersinia

Question 18

Purpose of GNR enrichment media

Answer 18

-Extend lag phase of normal flora and decrease lab phase of pathogens

Examples:
GN broth
Tetrathionate
Selenite-F broth

Question 19

EMB

Answer 19

Eosin Methylene Blue
-inhibits GP
–Holt-Harris (lactose + sucrose)
–Levine’s (sucrose only)

NLF = clear (except for sucrose fermenters P. vul and Serratia)
LF = black/purple metalic

Question 20

MAC

Answer 20

-More inhibitory than EMB
-Neutral red, crystal violet, bile salts
-Lactose

Question 21

SS Agar

Answer 21

-Neutral red, bile salts, brilliant green, sodium citrate
-H2S indicators = sodium thiosulfate + ferric citrate
-Lactose
–LF = red
–NLF = clear

Question 22

XLD agar

Answer 22

-Phenol red, deoxycholate, citrate
-Xylose, lactose, sucrose
-H2S indicator = ferric ammonium citrate + sodium thiosulfate

–Salmonella = red w/ black center
–Shigella = red

Question 23

HEK

Answer 23

-Bromothymol blue, acid fuchsin, bile salts
-Lactose, sucrose, salicin
-H2S indicator = ferric ammonium citrate + sodium thiosulfate

–Salmonella = blue-green w/ black center
–Shigella = clear/gree

-Proteus = yellow
-Other GNR = orange/pink

Question 24

BS Agar

Answer 24

-Bismuth Sulfite, dextrose, ferrous sulfate
-Best for S. typhi and lac+ Salmonella

-S. typhi = black colony w/ black zone and metallic sheen
-Other Salmonella = green

Question 25

Brilliant Green Agar

Answer 25

-Selective for Salmonella not typhi
-Phenol red, brilliant green, lactose

–Salmonella = white snowflake colony surround by red
–other colonies are greenish yellow
–S typhi and Shigella are inhibited

Question 26

Decarboxylase

Answer 26

-Enzyme that removes COOH from amino acidq
-Decarboxylase media, bromcresol purple, glucose, amino acid (lysine, ornithine, or arginine)
-Control tube w/o amino acid

1) Inoculate both tubes
2) Overlay both with mineral oil to prevent false alk at surface
3) Purple+ / Yellow=

Question 27

Phenylalanine deaminase

Answer 27

-Enzyme that removes NH2 from amino acid
-Phenylalanine agar

1) Inoculate and grow overnight
2) 10% ferric chloride
3) Green+

Proteus and Providencia are +++

Question 28

Urease

Answer 28

-Urea broth or agar w/ Phenol Red
-Hydrolysis of NH4OH

1) Inoculate and grow overnight
2) Red+ / Yellow=

Question 29

Gelatin

Answer 29

Broth w/ 12% gelatin
1) Stab gelatin and incubate up to 5 days
2) Refrigerate for 30 min before reading
3) Liquification+

Serratia, Proteus, Pseduo+
Hafnia, Kleb, Providencia=

Pseudomonas, some Serratia

Question 30

DNAase

Answer 30

1) Inoculate agar (methyl green) containing DNA and incubate
2) Add 0.1N HCl
3) Cloudy precipitate=; clearing around colony+

Question 31

Nitrate reduction

Answer 31

1) Inoculate Peptone broth or agar containing KNO3
2) Hold 5 days and test daily
3) Add Sulfanilic acid and alpha-naphthylamine to an aliquot
4) Red+
5) If neg, add Zinc to look for unreduced NO2