Gram-Negative Bacilli Flashcards
What specimen is used for laboratory diagnosis of Family Enterobacteriaceae and Related Enterobacteria ?
Urine, blood, pus, spinal fluid, sputum, or other material, as indicated by the localization of the disease process.
What is of unique significance in the gram staining of Klebsiella species ?
presence of large capsules, which can be observed as colorless to light pink halo around the bacilli
What is a useful alternative gram staining for Yersinia species ?
Wayson staining
What are the significant features shown by the Yersinia species under wayson staining ?
bipolar purple staining
central vacuole giving a characteristic “safety pin” appearance
What enrichment medium is best used in isolation of Salmonella ?
Selenite broth
What conditions must be met for Salmonella subcultures to be formed in selenite broth ?
8 - 12 hours
35°C
What is the reason for the specific conditions of Salmonella and selenite broth ?
coliforms or other intestinal flora may overgrow the pathogens within a few hours
What may happen if you overheat selenite broth ?
Overheating may produce a visible precipitate, making it unsatisfactory for use
What makes selenite broth a specific enrichment medium for Salmonella ?
It inhibits the growth of many species of gram-positive and gram-negative bacteria including the coliforms and many strains of Shigella
What is used as an enrichment medium for the recovery of Salmonella and Shigella from clinical and nonclinical specimens ?
GN (Hajna) broth
What conditions must be met for GN (Hajna) broth ?
4 - 6 hours
35°C
What is the reason for the specific conditions of the GN (Hajna) broth ?
Due to the relatively low concentration of desoxycholate, it is less inhibitory to E. coli and other coliforms
In the GN (Hajna) broth higher concentration of mannitol increases the growth of which Enterobacteriaceae species ?
Salmonella and Shigella
In the GN (Hajna) broth what does the sodium citrate and sodium desoxycholate do ?
inhibit gram-positive and some gram-negative bacteria
What broth is used as a selective enrichment for salmonella species ?
Tetrathionate broth with iodine-iodide solution
What makes tetrathionate broth a selective medium ?
The tetrathione which is formed in the medium by the addition of the iodine-iodide solution inhibits the normal intestinal flora of fecal specimens
What conditions must be met for the tetrathione broth ?
18 - 24 hours
35°C
What are things to take note of in preparation of tetrathione broth ?
After heating the broth base to boiling, add the iodine solution
No heating must be done after adding the iodine solution
What hemolytic signs are observed in E. coli on BAM ?
beta-hemolytic, many are shown from isolates of infections
What hemolytic signs are observed for Klebsiella pneumoniae on BAM ?
Non-hemolytic (gamma-hemolytic)
Significant characteristics of growth of Proteus on BAM ?
Swarming growth, distinct zonation
Culture usually gives off a “burnt gun powder” odor
What makes MAC agar a slightly selective medium for Enterobacteriaceae growth ?
The crystal violet dye and bile salts in the medium inhibit G+ bacteria and allow G- bacteria to grow
What change is there on MAC agar when a lactose fermenter is present ? and why ?
Bacteria that ferment lactose, the sole carbohydrate in the medium, produce acids, which, in the presence of neutral red as the pH indicator, results in the formation of pink to red colonies.
What is observed for non-lactose fermenters on MAC agar ?
Colourless colonies
What selective and differential medium is used for the detection of sorbitol-nonfermenting Escherichia coli serotype O157:H7 ?
MAC agar with sorbitol
What makes EMB agar a slgihtly selective medium for Enterobacteriaceae growth ?
Eosin Y and methylene blue inhibits growth of G+ bacteria
What is observed in lactose fermenting bacteria colonies on EMB agar ? and why ?
Lactose fermenting bacteria produce pink to purple colonies
due to the taking up of an eosin-methylene blue dye complex by the bacterial cells when the pH drops
Escherichia coli colonies show a characteristic green metallic sheen (GMS)
due to the rapid fermentation of lactose
What is observed in non-lactose fermenting bacteria colonies on EMB agar ?
Colourless or transparent
What makes SSA a moderately selective agar for Enterobacteriaceae growth ?
Bile salt, brilliant green, and citrates inhibit the growth of G+ bacteria
Detects lactose fermentation with the neutral red indicator forming pink or red colonies
Sodium thiosulfate is a source of sulfur. Any bacteria that produce hydrogen sulfide (H2S) are detected by the black precipitate formed with ferric citrate.
What are things to take note of in preparation of SSA ?
During preparation, heat to boiling to completely dissolve the agar. DO NOT OVERHEAT. DO NOT AUTOCLAVE.
What is observed in E. coli growth on SSA ?
pink to red colonies
What is observed in Salmonella or Proteus growth on SSA ?
colourless colonies with black centers
What is observed with Shigella growth on SSA ?
colourless colonies
What makes XLD agar a moderately selective agar for Enterobacteriaceae growth ?
Bile salt (sodium desoxycholate) inhibits G+ bacteria growth
Xylose, lactose, and sucrose are the fermentable carbohydrates, Xylose is fermented by practically all enterics except for the shigellae
Lysine decarboxylation by Salmonella, which initially produce yellow colonies due to xylose fermentation, results in delayed red colonies due to alkaline amines produced
Sodium thiosulfate and ferric ammonium citrate are the H2S indicator system. Formation of colonies with black centers.
What is observed with E. coli, Klebsiella, and Enterobacter growth on XLD agar ?
Yellow colonies and yellow conversion of the medium due to fermentation of carbohydrates
What is observed with salmonella growth on XLD agar ?
Red colonies surrounded by pink halo, the pink halo is due to the decarboxylation of lysine
There is also blackening of the colonies due to H2S production
What is observed with Proteus and Edwardsiella growth on XLD agar ?
Red colonies with black centers but no pink halo
What is observed with Shigella growth on XLD agar ?
Red colonies
What makes HEA agar a moderately selective agar for Enterobacteriaceae growth ?
Bile salt inhibit G+ bacteria growth and retard growth of many coliforms
Lactose, sucrose, and salicin are available carbohydrates for fermentation; acid fuschin reacts with bromthymol blue creating a yellow colour
Sodium thiosulfate source of sulfur and H2S is detected by ferric ammonium citrate
What is observed with E. coli growth on HEA agar ?
Yellow conversion of the medium
Yellow-orange to salmon-pink colonies
What is observed with Salmonella on HEA agar ?
Greenish-blue colonies with black centers
What is observed with Shigella on HEA agar ?
Faint green colourless colonies
What makes BSA a highly selective agar for Enterobacteriaceae growth ?
Isolating Salmonella typhi
Bismuth sulfite and brilliant green inhibit G+ bacteria growth and coliforms allowing Salmonella to grow
Bismuth sulfite is the source of sulfue and H2S is detected by ferrous sulfate, forming brown to black metallic sheen
What makes BGA a highly selective agar for Enterobacteriaceae growth ?
Isolation of Salmonella other than S. typhi/S. paratyphi
Brilliant green dye inhibits growth of G+ bacteria growth and most of G-
Phenol red is indicator for fermentation of lactose and/or sucrose
What is observed with Salmonella species other than S. typhi and S. paratyphi growth on BGA ?
White to red colonies surrounded by red zones
What is observed with S. typhi and S. paratyphi growth on BGA ?
No traces of growth
In the inoculation and incubation of the specimen what is to be done if negative after incubation of 24 hours ?
Reincubate for an additional 24 hours and select suspicious colonies for definitive biochemical or serologic testing
In preparation of Yersinia enterocolitica what is the enrichment process ?
Cold enrichment of feces by 4°C for 1-3 weeks in buffered saline to enhance Y. enterocolitica recovery
What is observed with Y. enterocolitica growth on MAC ?
Lactose-negative colonies , flat, colourless, or pale pink
What is observed with Y. enterocolitica growth on CIN agar ?
Fermentation of mannitol in the presence of neutral red results in a characteristic “bull’s-eye” colony, colorless with red center
What makes CIN agar a good medium for detection of Y. enterocolitica ?
Crystal violet, sodium desoxycholate and Irgasan (triclosan) inhibits growth of G+ bacteria and G- bacteria
Supplementation with cefsulodin and novobiocin improves inhibition of normal enteric bacteria.
What makes TSI nutritionally rich ?
incorporation of four protein derivatives—beef extract, yeast extract, peptone, and proteose peptone
What fastidious bacteria specie is excluded in TSI growth ?
obligate anerobe
What is the difference between the two chambers in the TSI tube ?
upper slant is aerobic
bottom slant is anaerobic
What colour indicates an acid slant or butt in TSI agar ?
yellow
In the result of an alkaline slant and acid butt what causes the change of the slant ?
the release of amines to counteract the small percentage of acid
What showcases gas production in TSI ?
Presence of cracks in the medium or the “pulling away” of the medium from the walls of the test tube
Where is hydrogen sulfide production first seen in the TSI tube ?
The butt because it becomes acidic with glucose fermentation
How is inoculation done on TSI ?
First stab the butt 2-3 mm deep
Then stroke the slant from bottom to top in back forward motion
Why must the screw caps be loosened slightly during incubation for TSI ?
to promote typical reactions
How will alkaline slant, acid butt, with gas, and H2S production be presented if shown in the TSI ?
K/Ag + H2S
What is the difference between KIA and TSI ?
KIA lacks sucrose and the H2S indicator is ferric ammonium citrate
What does the IMViC reactions stand for ?
Indole test
Methyl red test
Voges-Proskauer test
Citrate utilization test
What is the incubation period for MR-VP broth ?
2 - 4 days
What must be taken note of when inoculating a series of tubes of differential culture media with an unknown organism for the IMViC reactions ?
the citrate medium be streaked first to prevent carryover of proteins or carbohydrates from the other media
What must be taken note of when using the ehrlich reagent in the indole test ?
Step should be preceded with the addition of 1ml of xylene or chloroform, because indole is soluble in organic compounds
What is the positive result of the indole test ?
bright fuschia red colour at interface of the reagent and the broth
What is the negative result of the indole test ?
yellow colour or no change
In the MR test, because other organisms may produce smaller quantities of acid from the test substrate, an intermediate orange color between yellow and red may develop. What is the result ?
This does not indicate a positive test
What is the right order of addition of reagents in the VP test ?
alpha-naphthol and then 40% KOH
What is the purpose of alpha-naphthol in the VP test ?
serves as a catalyst to bring out a red complex
After shaking the tube for exposure of the VP test to oxygenic atmosphere how long must it be left undisturbed for ?
10 - 15 minutes
How many drops of p-dimethylaminobenzaldehyde containing solutions must be added to the indole test ?
15 drops
How many drops of methyl red indicator must be added to the MR test ?
5 drops
Why must the VP test not be read for standing over an hour ?
negative VP cultures may produce a copper-like color, potentially resulting in a false-positive interpretation
What is the positive and negative result for citrate utilization test ?
\+ = prussian blue or deep colour blue - = retention of green colour
How is motility test possible with SIM agar ?
semisolid nature of the medium
What is the correct order of observation of results in the SIM test ?
Motility first and then sulfide before addition of reagents for indole detection because reagent may diffuse into the medium making it hazy or cloudy which may result in false motility interpretation
What other media is available for motility testing ?
Motilility Test Medium
Motility Indole Ornithine (MIO) Medium
Motility Indole Lysine Sulfide (MILS) Medium
MIL (Motility-Indole-Lysine) Medium
Lysine decarboxylation by bacteria produces what alkaline reacting amine ?
cadaverine
What is the positive and negative result for lysine decarboxylation ?
\+ = purple butt - = yellow butt
What is the positive and negative result for lysine deamination ?
\+ = red slant - = purple slant
What are the three amino acids routinely tested for the identification of Enterobacteriaceae ?
Lysine, ornithine, and arginine
What is the positive and negative result for the urease test ?
\+ = pink red - = yellow
What is done after incubation in PAD tests ?
add 4 or 5 drops of the ferric chloride directly to the surface of the agar
As the reagent is added, the tube is rotated to dislodge the surface colonies
What is the positive and negative result for the PAD test ?
\+ = green - = not green
What does ONPG mean ?
o-nitrophyl-β-D-galactoyranoside
What is the visual evidence of ONPG going through hydrolysis ?
ONPG is a colorless compound and after it cleaves into o-nitrophenol it becomes yellow
What are the two enzymes required for the production of acid in the lactose fermentation test ?
lactose permease and β-galactosidase
What enzyme of the lactose fermentation test is required for the lactose molecule to penetrate the bacterial cell ?
Lactose permease
What enzyme of the lactose fermentation test is required to cleave the galactoside bond, producing glucose and galactose ?
β-galactosidase
What considerations are there when reporting results for ONPG test ?
visible yellow color reaction within 5–10 minutes
Most tests are positive within 1 hour; however, reactions should not be interpreted as negative before 24 hours of incubation
What enzyme is part of the MUG test ?
β-glucuronidase
What is the result for MUG test of E. coli serotype O157:H7 ?
negative
