LAB 8 - Enterobacteriaceae Flashcards
aerogenic vs anaerogenic
organisms that ferment glucose producing acid and gas vs acid only
these organisms cause GI distress due to toxins
B. cereus
S. aureus
C. perfringens
Wilson Blair agar
inhibits GPOS
what is the general rule for inoculation of media and biochemical tests?
least selective (most enriched) to most selective
liquid to semi solid to solid
E. coli on SMAC
white
Yersinia on HEK
tgn/yel
E. coli O157:H7 on STEC chromagar
mauve
Yersinia on CIN
bulls eye
spot indole
p-dimethylamino-cinnanaldehyde
highly sensitive but will not work on pigmented organisms
Kovac’s indole vs spot indole
Kovac’s = paradimethylaminobenzaldehyde; cherry red pr pink
spot indole = blue/green
tests for tryptophanase
what is the purpose of aerosporin media?
has polymyxin B (antibiotic for GNBs)
inhibits GNB; selective for GPS
phenylethyl alcohol agar (PEA)
- The active ingredient, phenylethyl alcohol, inhibits or markedly reduces the growth of Gram-negative organisms by interfering with DNA synthesis
- supports growth of GPCs and anaerobes
colistin nalidixic acid
Colistin, a polypeptide antibiotic of the polymyxin group, and nalidixic acid, a first-generation quinolone, are primarily active against gram-negative bacteria thereby making Columbia CNA Agar a good medium for the selective isolation of gram-positive cocci.
decarboxylase test
- after the fermentation of glucose in the medium, acids are produced which lower the pH = purple to yellow
- if the organism produces decarboxylase, decarboxylation, or hydrolysis of the amino acid occurs in response to the acid pH = alkaline end products (amies) = purple
- Decarboxylation is an anaerobic reaction
pH indicators of decarboxylase test
bromcresol purple and cresol red
indicator for TSI
phenol red
when is susceptibility testing performed on enteric pathogens?
self-limiting so usually don’t
- unless immunocompromised, hospitalized pts, prolonged infections
- Shigella and Vibrio = automatic testing due to severity
serological methods for ID of Shigella, Salmonella, and E. coli O157:H7
- O (somatic antigen) - heat-stabile antigen located in cell wall, resistant to alcohol
- H (flagellar) - heat-labile; resists formalin
- K (capsular) - heat-labile polysaccharide; K1 of E. coli, Vi of S. Typhi; may mask O antigen
Serologic grouping of Shigella sp. is based on the ____ antigen
O antigen
- if agg fails = suspension is heated to remove capsular antigen and is retested with O antisera
Shigella serotypes
A = S. dysenteriae
B = S. flexneri
C = S. boydii
D = S. sonnei
primary intestinal pathogens
- Salmonella, Shigella, Yersinia enterocolitica, E. coli O157:H7 = true pathogens
- NOT commensal flora of GIq
oxidase test principle
- tetramethyl-p-phenylenediamine (TMPD) interacts with the ETC at level of cytochrome c
- produces dark purple indophenol
- found only in bacteria whose electron transport systems contain cytochrome c oxidase
what can inhibit oxidase activity?
carb fermentation or components of selective media
why must TSI reactions be read within the 18-24 hr inc period?
acid reactions may revert to alkaline due to oxidative decarboxylation; glucose fermenters may falsely appear to be non-fermenters
detection of hydrogen sulfide in TSI
due to sodium thiosulfate and ferrous ammonium sulfate
TSI ingredients
pH-sensitive dye (phenol red), 1% lactose, 1% sucrose, 0.1% glucose, as well as sodium thiosulfate and ferrous sulfate or ferrous ammonium sulfate
citrate utilization
- certain organisms can use citrate as their sole source of carbon
- pos = growth -> blue due to alkaline end products produced (bromothymol blue)
- useful for differentiation of lactose fermenters
- Kleb and Enterobacter utilize citrate but E. coli does not
Malonate utilization
- utilize malonate as a source of carbon?
- end products = acetate and CO2
- alkaline pH so bromthymol blue changes from green to blue (alkaline) in a positive test
Phenylalanine deaminase test
- test for ability to deaminate phenylalanine to phenyl pyruvic acid by adding ferric chloride as an indicator
- Proteus-Providence (+); rest of colifs (-)
- green is pos
urease
- splits urea into CO2 and ammonia
- phenol red turns deep cerise due to alkalinity
Orthonitrophenyl-beta-galactopyranoside (ONPG) test
- orthonitrophenyl-beta-galactopyranoside
- detects beta-galactosidase (cleaves lactose into glucose & galactose)
- also depends on PERMEASE ( transports lactose into cells)
- yellow is pos
Lactose fermenter (ONPG pos)
both permease enzyme and beta galactosidase
E. coli, Kleb spp., Enterobacter spp
Late lactose fermenter (ONPG pos)
only beta galactosidase
Citrobacter spp, Arizona sp
Y. enterocolitica motility
+ in RT
- in vivo
MAC ingredients
Peptone
Lactose monohydrate
Bile salts (and CV inhibit GPC)
Sodium chloride
Neutral red
Crystal Violet
SMAC
E. coli is pink on MAC and pk/ng on SMAC
whereas E. coli O157:H7 is pk on MAc and wt on SMAC
