Micro lab quiz 5

catalase test

Assayed: To determine if organisms contain catalase to breakdown harmful by products (H2O2) produced during aerobic respiration. Perform: Hydrogen peroxide is added to a culture after being incubated . Interpretation: Bubbles= catalase is present No bubbles= catalase is absent

BHIA stab tube

Assayed: oxygen requirement/tolerance of a species Perform: tubes of a solid medium inoculated by stabbing with inoculating needle and then incubating, then seeing results for growth along stab line Interpretation: Oxygen species are usually coagulase positive

Voges-Proskaur test

Assayed: tests for neutral end products of glucose fermentation (acetoin) Perform: the broth is incubated and then VP reagents are added. Interpretation: -Red= 2,3 - butanediol fermentation (acetoin produced) -No color change= no 2,3 - butanediol (acetoin not produced)

SIM tube

Assayed: sulfur reduction, indole production from tryptophan, and motility Perform: Medium is inoculated with a single stab from inoculating needle, then incubated. ( Kovacs reagent is added after incubation for indole results) Interpretation: Sulfur Reduction: -Black in medium= sulfur reduction (salmonella) No black= sulfur isn't reduced Indole production: -Red in alcohol top layer of Kovacs reagent= tryptophanase is present -No color Change= tryptophanase not present Motility: -Growth outward from stab line= motility -No growth= nonmotile -Blackened medium= Undetermined

S. aureus

Coagulase + Causes clotting Common HAI

S. epidermis

Coagulase - HAI, UTIs, other infections

S. saprophyticus

Coagulase - Most common cause of UTIs

EMB agar

-Complex (chemically undefined) selective for gram negatives and differential from lactose fermenters and non lactose fermenters -Used for isolation of fecal coliforms Ingredients: -Gelatin provides organic carbon and nitrogen -Lactose is fermented by acid end products by coliforms such as E. coli and enterobacteriacae, not pathogens like shigella or salmonella (differential) -Eosin y and Methyl blue inhibit growth of most gram positive organisms and react with vigorous lactose fermenters whose acid end products turn growth dark purple-black (selective) Results: -Poor/no growth: organism is inhibited by eosin or methyl blue = gram + -Good growth: organism is not inhibited by eosin or methyl blue = gram - -Growth is pink: organism ferments lactose or sucrose with acid production = possible coliform (if green metallic then it is most likely e. coli) -Colorless growth: organism does not ferment lactose = noncoliform

HE agar

-is a complex (chemically undefined), moderately selective for gram negatives, and differential between coliform. Medium is used to isolate and differentiate salmonella and shigella species from other gram negative enteric organisms Ingredients: -Test is based on ability to ferment lactose, sucrose, or salicin to acid end products and to reduce sulfur to hydrogen sulfide gas. Ferric ammonium citrate is included as a source of oxidized iron to react with any H2S produced to form the black precipitate ferrous sulfide. Bile salts are included to prevent or inhibit growth of gram positive organism. The bile salts also have a moderate inhibitory effect on enterics, so relatively high concentrations of animal tissue and yeast extract are included to offset this. Differentiation is possible as a result of the various colors produced in the colonies and in the agar. Results: -Blue green growth with black precipitate: organism doesn't ferment lactose, sucrose but reduces sulfur to H2S- probably salmonella -Blue-green growth no black: Organism doesn't ferment lactose, sucrose, or reduce sulfur- probably shigella/rare salmonella -Pink/orange growth: organism produces acid from lactose or sucrose-not shigella or salmonella -Poor/no growth: organism inhibited by bile or one of the dyes (gram +) -Good growth: organism isn't inhibited by bile or one of the dyes (gram -)

BHIA stab

1-4.) facultative anaerobe

How is MacConkey selective

MacConkey's is a selective medium that inhibits the growth of Gram-positive bacteria due to the presence of crystal violet and bile salts. Gram-negative bacteria grow well on MAC.

What does MSA stand for?

Mannitol salt agar

what is BHIA stab tube purpose

Oxygen requirements of tubes -Aerobic respiration

How is MSA selective

This medium only grows salt-loving bacteria Mannitol Salt is a selective bacterial growth medium because it has a very high concentration of NaCl (7.5%). Most bacteria cannot survive in this highly saline, hypertonic environment. But the genus Staphylococcus has a protective slime layerthat protects it in a harsh, salty environment. So Staph grow well in this media.

Blood agar

Used for isolation of many types of bacteria. also used to differentiate bacteria based on their hemolytic characteristics, especially for determining hemolytic ability of gram positive cocci, typically strep species. Several types of gram + coli produce exotoxins called hemolysins, which are able to destroy RBC's and hemoglobin. Ingredients: 5% sheep blood in tryptic soy agar base that allows for differentiation of bacteria based on ability to hemolyze RBC Results: -Clearing around growth: organism hemolyzes RBC completely B-hemolysis (S. pyogenes) -Green around growth: Organism hemolyzes RBC partially - A-hemolysis (S. pneumoniae) -No change: Organism does not hemolyze RBC - Y-hemolysis (S. epidermis)

Mannitol salt agar

Used for isolation/differentiation of staphylococcus aureus from other staph species. Ingredients: -Selective: NaCl makes the medium selective because its concentration is high enough to dehydrate and kill most bacteria -Differential: mannitol provides the substrate for fermentation and makes the medium differential -pH indicator phenol red: indicates whether fermentation with an acid end product has taken place by changing color as pH changes -Yellow below pH 6.8 -Red at pH 7.4-8.4 -Pink at pH 8.4 and above Results: -Poor/no growth: means the organism is inhibited by NaCal (not staph) -Good growth: organisms is not inhibited by NaCl (staph) -Yellow growth or halo: organism produces acid from mannitol fermentation -Red growth: organism doesn't ferment mannitol (is a staph other than staph aureus)

S. pneumonia

alpha hemolytic found in throat most common cause of bacterial pneumonia

How is MSA differential

bacteria that grow on MSA that can ferment mannitol, a sugar alcohol, will turn the medium from its original pink color to a bright, neon yellow. This growing medium is also differential because it contains a dye that identifies types of Staphylococcus that produce an organic acids from mannitol fermentation (eating mannitol, a type of alcohol). Pathogenic Staph, such as Staphylococcus aureus, are mannitol fermenters, and when growing on Mannitol Salt Agar, their wastes turn the MSA a bright yellow color.

Coagulase

differential for staph aureus only

what does differential mean?

differential medium is a growth medium that allows two or more organisms to be distinguished from one another by some parameter, such as growth vs. no-growth, distinguishing colors, distinguishing morphologies, etc.

How is EMB differential

used for the isolation and differentiation of coliforms and fecal coliform. Differentiation between these gram negative bacilli is based on the colony color. Colored colonies in Eosin Methylene Blue (EMB) Agar: Lactose fermenter Colorless colonies in Eosin Methylene Blue (EMB) Agar: Non lactose fermenter The combination of the two dyes eosin and methylene blue inhibits most Gram positive bacteria but allows many Gram negative organisms to grow.

MRVP

1.) MR+ VP- 2.) MR+ VP- 3.)MR+ VP- 4.) MR+ VP+

strategy to identify unknown bacteria

1.) Mixed sample (body sample aka skin, fecal, urine) - isolation streak plate 2.) pure culture 3.) gram stain to determine cellular morphology 4.) carry out specialized tests (endospore stain, acid fast, banana broth) 5.) Physiological/biochemical/metabolic tests (selective and differential media) 6.) Dichotomous key (bergeys)

SIM

1.) Yes No enzyme (indole -) motile 2.) No no enzyme motile 3.) No indole + motile 4.) No no enzyme non motile

Durham fermentation tubes

1.) red cap= A green cap= NR blue cap= NR 2.) NR NR NR 3.) AG AG NR 4.) AG AG NR

Durham fermentation tubes

Assayed: Ability of organism to ferment glucose, lactose, and mannitol and produce acid or gas. Detection based on pH changes and trapping gas in Durham tube. Perform: a smaller Durham test tube inserted upside down in Phenol Red Broth. This small tube is initially filled with the solution with a carb in which the microorganism is to be grown. Interpretation: -Yellow broth, bubble in tube= Fermentation with acid and gas end products pH<6.8 -Yellow Broth, no bubble in tube= Fermentation with acid end products, no gas pH<6.8 -Red broth, no bubble in tube= No fermentation pH in between 6.8 and 7.4 -Pink Broth, no bubble in tube= Degradation of peptone, alkaline end products pH>7.4

Methyl red test

Assayed: Detect organisms capable of performing mixed acid fermentation Perform: the broth is incubated and then drops of methyl red indictor dye are added. Red at pH 4.4 Yellow at 6.2 , mixed in-between Interpretation: -Red= mixed acid fermentation -No color change= no mixed acid fermentation

How is HE selective

Bile salts and the dyes bromthymol blue and acid fuchsin inihibit the growth of most Gram positive organisms.

Hemolysis

Breakdown of RBCs tested with blood agar plates (selective for streptococcus species) Hemolysis: break down of RBC (gram + cocci) -Selective for streptococcus Differentiates: B- hemolysis (complete): streptococcus pyogenes or S. agalctiae = bacitrin resistant A-hemolysis (partial) = S. pneumonia G-hemolysis = none

Selective and differential tests, how are they metabolic?

Breakdown of carbon subunits to make ATP? -Glucose broken into CO2 and water in aerobic cellular respiration using an ETC that contains enzymes like oxidase -Sugars (glucose) or pyruvate into fermentation products like acid Breakdown of macromolecules -Exoenzymes exit the cell and break large substrates into smaller substances: like amylase

what does bubbles indicate

Catalase postive (aka produces catalase) -Aerobic respiration

Citrate agar

Differentiate members of enterobacteriacae (anaerobes), tells us the ability to use citrate as sole carbon source and perform citrate fermentation. Ingredients: -Sodium citrate- source provides bacterial species that posses enzyme citrate permeate to transport citrate into cell and ferment it -Ammonium phosphate- nitrogen source, must be able to survive with ammonium in this form -Bromothyl blue dye: as indicator as pH goes up Results: -Blue: citrate is utilized -No color change with growth: citrate utilized -No color change with no growth: citrate not utilized -Citrate positive bacteria hydrolyze citrate into oxaloacetate and acetate using enzyme citrate lyase. Oxalacetate is covered to pyruvate.

Bacitracin sensitivity

Distinguish streptococcus pyogenes from other strep bacteria S. pyogenes is sensitive to bacitracin and others are resistant to it

What does EMB stand for

Eosin Methylene Blue agar

Milk agar

Exoenzyme assayed: caseinase -Clearing in agar: present -No clearing in agar: absent Substrate= casein (Milk protein) Product= peptides and amino acids

Catalase

Exoenzyme assayed: catalase adding hydrogen peroxide -Bubbles: catalase present -No bubbles: catalase is absent Substrate: hydrogen peroxide End product: water and oxygen

Coagulase

Exoenzyme: coagulase -Coagulation in agar: present -No clearing in agar: absent Substrate= plasma fibrinogen Product= fibrin

S. agalactiae

Found in vagine neonatal infections bacitracin resistant known as group B strep

Lance field classification

Grouping catalase-negative bacteria based on the carbohydrate composition of antigens found on their cell walls

what does HE stand for

Hektoen Enteric Agar

How is EMB selective

It is selective culture medium for gram-negative bacteria (selects against gram positive bacteria)

How is HE differential

Lactose-fermenting organisms such as E. coli produce a black precipitate on EMB. Their colonies will be either black or possess dark centers with transparent, colorless peripheries. Non-lactose fermenters such as Proteus sp., Salmonella sp., or Shigella sp. appear pink or uncolored. Thus, the medium is considered differential with respect to lactose fermentation.

MacConkey agar

Selective for gram negative, differentiates members of the enterobacteriaeceae based on the ability to ferment lactose. Ingredients: -Selective ingredients: Bile salts, crystal violet-inhibit growth of gram + bacteria -Neutral red dye is a pH indicator -Differential: lactose provides the substrate for fermentation and makes the medium differential Results: -Poor/no growth: Organism gram + -Good growth: Gram - -Pink/red growth: organism produces acid from lactose fermentation (possible coliform) -Colorless growth: organism does not ferment lactose (noncoliform)

what does selective mean?

Selective media is a type of media that selects for the growth of one type of organism, while inhibiting the growth of others

S. pyogenes

Skin infections, strep throat, known as group A strep

SIM tube

Sulfur reduction- to determine if organisms can reduce sulfate at the ETC (anaerobic), black precipitate Indole- indicates E. coli Motility- when positive it indicates salmonella and E. coli, motile -Anaerobic respiration

How is MacConkey differential

can differentiate those Gram- bacteria that are able to ferment lactose. With MacConkey's, it is not the media that changes color, but rather the actual colonies of lactose fermenting bacteria that appear pink. Non-lactose fermenting bacteria will be colorless (or, if the colonies have any color, it will be their natural color rather than pink).

MSA

selectie for staph species (high salt) and differential for mannitol fermentation

Catalase test

selective for staph species Negative- strepto species then if MSA is positive = staph