MLTS 2061 - Exam 1

ONPG

• test considered to be very sensitive for lactose-fermentation

• changed to Ortho nitrophenol by beta-galactosidase

• can be disc or tube test

• pos. = yellow = enzyme B-galactosidase

What can the ONPG test be helpful to differentiate?

• Citrobacter (pos.)

• Salmonella (neg.)

H₂S Production

• organism that possess H₂S producing enzymes release sulfur from sulfur-containing compounds to produce colorless H₂S gas

• H₂S reacts w/ iron salt to form ferrous sulfide

• pos. = black precipitate

What two organisms can H2S be helpful to differentiate?

• Salmonella (H2S+)

• Shigella (H2S=)

What three medias show visible H2S production?

1. HE

2. XLD

3. TSI

Triple Sugar Iron Agar (TSI Slant)

• sugar fermentation leads to acid production and change in color of pH indicator

• semi-solid media having a slant and bu-tt containing: 3 sugars (lactose, sucrose, glucose) and iron

In triple sugar iron agar, what does each color indicate?

• yellow = acid (A) fermentation has occurred

• pink = alkaline (K) fermentation has not occurred

In triple sugar iron agar, what does each DEEP color indicate?

• pink deep = glucose not fermented

• yellow deep = glucose fermentated

In triple sugar iron agar, what does each colored SLANT indicate?

• pink slant = lactose/sucrose not fermented

• yellow slant = lactose and or sucrose are fermented

In triple sugar iron agar, what are do other reactions indicate?

• black precipitate = H2S production

• bubbles/cracks = gas production

What are some things to be mindful when performing a TSI test?

• record: slant / deep

• stab the butt and streak the slant

• very important to leave cap loose

Oxidase

• tetramethyl-para-phenylene-diamine dihydrochloride reacts with/ cytochrome C to produce blue/purple color

• pos. = blue or purple

What is the oxides test helpful for?

• differentiate Enterobacteriaceae from other non-fermenters

• Enterobacteriaceae family = oxidase neg.

• Pseudomonas spp = oxidase pos.

Nitrate Reduction

• if an organism reduces nitrates to nitrates, a red color develops when sulfasilic acid and N,N-dimethyl-l-l-naphthylamine are added

• pos. = red (nitrate to nitrite)

• no color, add zinc dust = clear (pos.); red = neg.

What does no color after the addition of zinc dust mean in a nitrate reduction test?

nitrates were reduced to N2 or NO2, meaning pos. nitrate reduction

Carbohydrate Fermentation

• when a carbohydrate is fermented, the production of acidic end products causes a color change in the pH indicator

• phenol red indicator → changes from red to yellow w/ carbohydrate fermentation

• pos. = yellow

• all Enterobacteriaceae ferment glucose

What are some examples of carbs/sugars?

• glucose

• lactose

• sucrose

• mannose

• sorbitol

• mannitol

• xylose

Indole Tube Method

• enzyme tryptophanase dominates tryptophan w/ production of indole

• tryptone broth is inoculated with/ organism—rich source of AA tryptophan

• addition of Kovac’s reagent (paradimethylaminobenzaldehyde) forms pink colored complex w/ indole

• pos. = pink

What colonies from what media should be used for the indole tube method?

BAP or Choc agar, not MAC

What three procedures can be used to perform the spot indole test?

• filter paper method

• swab method

• direct agar plate method

Explain the filter paper method for the spot indole test.

• moisten a strip of filter paper w/ the indole reagent

• using a sterile inoculating loop, pick up fresh (18-24 hrs.) colonies grown in a tryptophan-rich medium and rub the colonies over the moistened filter paper

• observe for color change within 20 secs.

Explain the swab method for the spot indole test.

• using a sterile swab, pick up some fresh (18-24 hrs.) colonies of sample bacteria grown in a tryptophan-rich medium

• add a drop of indole reagent directly over the colonies

• observe for color change within 20 secs.

Explain the direct agar plate method for the spot indole test.

• drop indole reagent directly over the fresh colonies of sample grown in tryptophan-rich medium

• observe the color change on the colonies and surrounding medium within 20 secs.

• pos. = blue

Methyl Red

• principle: if glucose is metabolized by the mixed acid fermentation pathway, acidic end products are produced, resulting in low pH

• pos. = red after addition indicator

• pH < 4.4 = indicates carb fermentation

Voges-Proskauer (VP)

• principle: acetoin is produced from an alternate pathway for glucose medium. End product: acetoin

• pos. = red when potassium hydroxide (KOH) and alpha-naphthol are added

Citrate

• principle: if an organism can use citrate as its sole source of carbon, the pH increases and the pH indicator changes color

• pos. = green → blue

Urease (Christensen’s Urea Agar)

• principle: enzyme urease breaks down urea. Ammonia released and pH increases. Change in color or pH indicator

• pos. = yellow → pink

Phenylalanine Deaminase (PD; PAD)

• principle: PD dominates phenylalanine to phenylpyruvic acid. Phenylpyruvic acid reacts w/ ferric chloride to produce a green color

• pos. = after addition of 10% ferric chloride → green

What enterobacteriaceae species are PD positive?

• Proteus

• Providencia

• Morganella

What controls are used for PD?

• pos. = Proteus

• neg. = E. coli

Motility

• principle: motile organisms grow away from the stab line in medium w/ an agar concentration of 0.4% or less

• pos. = movement away from the stab line or a hazy appearance throughout the medium after overnight incubation

What Enterobacteriaceae species are non-motile?

• Klebsiella

• Shigella

• Yersinia (at 22℃)

What Enterobacteriaceae species are used as controls for motility?

• pos. = E. coli

• neg. = Klebsiella

MIO (Motility, Indole, Ornithine)

• pos. motility = turbidity or cloudy growth extends from the line of inoculation

• pos. indole = pink to red color band at the top of the medium after addition of Kovacs reagent; neg. = yellow

• pos. ornithine = dark, turbid purple; neg. = yellow

Hektoen Enteric (HE) Agar

• selective and differential agar primarily used to recover Salmonella and Shigella

• contains indicators of lactose fermentation and hydrogen sulfide production

• inhibitors to prevent growth of gram-pos. bacteria

What would Salmonella and Shigella look like on the HE agar?

• Salmonella = black colonies

• Shigella = green colonies

Xylose Lysine Deoxycholate (XLD) Agar

• selective growth medium used in the isolation of Salmonella and Shigella

• pH of 7.4, leaving a bright pink or red appearance due to the indicator phenol red

• contains indicators of lactose fermentation and hydrogen sulfide production

• inhibitors for gram pos. bacteria

What would Salmonella and Shigella look like on the XLD agar?

• Salmonella = black colonies

• Shigella = red colonies

MacConkey Agar

• selective and differential to isolate gram-neg. and enteric bacilli and differentiate them based on lactose

• LF = pink; NLF = clear

MacConkey with Sorbitol (Sugar)

• variant of MAC used in the detection of E. coli O157:H7

• E.coli O157:H7 = clear (does not ferment sorbitol)

EMB (Eosin Methylene Blue Agar)

E. coli produces a green metallic sheen

IMViC

• group of individual tests used in microbiology lab testing to identify and organism in the coliform group

• useful in distinguishing members of the Enterobacteriaceae

Coliform

• gram neg. aerobic or facultative anaerobic rod

• produces gas from lactose within 48 hrs.

• presence indicates fecal contamination