Lab material Flashcards
What color are Micrococcus luteus colonies?
yellow
What are hemolysins?
- extracellular enzymes produced by bacteria that are capable of lysing RBC
- used in the classification of streptococci
What is the Beta hemolytic group?
- the bacteria that form a soluble hemolytic substance capable of completely lysing RBC
- Colonies will be surrounded by a zone of complete hemolysis
What is the Alpha hemolytic group?
- bacteria that produce a green discoloration of the agar in the immediate vicinity of the colongy
- The greening is followed by a partial lysis of the cells in the area
- Lysis is supposedly due to hemolyzing concentrations of acid
What is a general pupose medium?
- The exact chemical composition is unknown, and can vary from batch to batch.
- Most bacteria can grow on it
- Ex:
- Blood agar
What is a differential medium?
- allows differentiation of bacterial species or types based on phenotypic characteristics
- use pH indicators to siignify whether bacteria degrade nigtrogenous compounds or charbohydrates
- Indicators:
- Phenol Red
- Bromothymol Blue
- Bromocresol purple
- Methy Red
- Neutral Red
- Litmus
What is a selective media?
- Used for the isolatin of specifc types of bactria
- contains a chemical that inhibits growth of a specific group of bacteria
- May also be a differential media
What is the CAMP test?
- Christie, Atkins, andMunch-Peterson
- Used to identify group B streptococci
- S. aureus produces B-toxin whose hemolytic activity is enhanced by an exracellular factor produced by group B strptococci
- Test is made by making a single streak of S. aureus down a blood agar plate, and then placing a single streak of the group B Streptococcus suspect perpedicular to it. Must NOT touch, but be very close. Then incubate at 37C
- Positive if complete hemolysis develops
What is the Carbohydrate Fermentation Test?
- Conducted with Phenol Red Broth
- Test if a particular organism will or will not produce acid from the particulr carbohydrate present in the medium
- variety of medium compositions
- Add: Glucose, Maltose, Mannitol, Trehalose (more than 50 different ones)
- Inoculate with a small amount of bacteria from a medium tht does not contain other fermentable carbhydrates (Ex: MAC)
- Incubate 37C 24h
- Red/Pink = Negative
- Yellow = Acid production = Positive
What is the Catalase Test?*
- identifies bacteria that produce the enzyme catalase and are able to degrade hydrogen peroxide
- H2O2 produced by aeroves, facultative anaerobes, and microaerophiles during oxidative metabolism of carbohydrates
- Highly toxic to the cell
- Differentiates between the following genera:
- Staphylococcus (+) from Streptococus (-)
- Bacillus (+) and Clostridium (-)
- Listeria / Corynebacterium (+) from Erysipelothrix (-)
- Most G- are Catalase +
- Place Hydrogen Peroxide on a slide and add inoculum. Observe for bubbles
- Immediate bubbles = Positive
- No bubbling = Negative
Blood Agar colonies can cause a false positive test, due to RBCs containing catalase
What is Chocolate Agar?
- Contains the same ingredients as blood agar
- RBC are added at 85C to partiallly lyse them and release hemin (x factor) and NAD (V factor) which are required for some gastidious bacteria
What are Citrate Agar Slants?
- Used to deterime if an organism is capable of utilizing citrate as the sole source of carbon for metabolism with a resulting alkalinity
- Inoculate slant by streaking, icubate 37C for 24-48h
- Changes to Blue = Acetate production = Positve
- Stays Green = Negative
- Selective and differential
What is the Gram Stain?
- Categoizes bacteria into 2 broad groups:
- Gram + (blue-purple)
- Gram - (pink -red)
- Gram stains work by creating a large I2-Crystal Violet complex inside the cell
- The alcohol is dissolves lipids in the outer membrane of Gram - baceria, allowing the I2-Crystal Violet complex to leak out of the cell.
- Gram + have thick proteoglycan and polysaccharide membranes, no lipid = no leakage
- The safranin is then added and colors only the the G- cell that were decolorized pink
- The alcohol is dissolves lipids in the outer membrane of Gram - baceria, allowing the I2-Crystal Violet complex to leak out of the cell.
What is Gram Variable?
- Gram + organism staining Gram -
- Treatments can disrupt the cell wall of Gram+ organisms (Ex: lysozyme, autolytic enzymes, growth conditions, age) and allow leakage of teh I2-Crystal Violet complexes causeing Gram + organisms to stain Gram -
- Gram + cells may appear Gram - after antibiotic therapy
How do you perform a Gram Stain?
- Place inoculum on slide
- Broth samples just need a loopful of medium
- Plate samples need to be added to a drop of water and mixed thoroughly
- Air dry at room temp
- if liquid is present during heat-fixing the cells will distort or lift off the slide
- Heat fix
- pass slide through the flame to adhere the cells to the slide
- Warm, NOT hot - will distort normal shape and structure
- Gram stain:
- Flood slide with Crystal Violet for 1 minute
- Drain and rinse with tap water
- Flood slide wwith Iodine for 1-2 minutes
- Rinse with tap water
- Decolorize with 95% ethyl alcohol (use dropper) until stain is no longer bein eluted from the smear
- Rinse with tap water
- Flod slide with safranin for 1 minute
- Rinse with tap water, air dry (or blot, but do not rub)
- Examin under oil-immersion
What is the Indole test?
- Determines if a given species of baceria possesses the enzyme tryptophanase
- Will deaminate the AA tryptophane to produce indole and possibly skatole
- Medium must contain tryptophan - (Ex Tryptone Broth 1%)
- Inoculate the tube with a generoush sample.
- Incubate 37C for 24-48 h
- Add 5 drops of Kovac’ reagent
- Red ring = Positive
- No red = Negative
Why does Serratia marcescens cause a false positive Indole test?
It’s red pigment extracts into the Kovacs’ solution and give it a reddish appearnce and constitutes a false positive
What is MacConkey Agar
- Growth medium of bile salts and crystal violet
- inhibit most Gram positive and some gram negative bacteria
-
Neutral red is pH indicator
- Lactose fermentors will become pink
- Can lose color due to changes in the A/B balance
- Differentia and Selective
What is Mannitorl Salt Agar?
- Selective medium to isolate coagulase-positive staphylococci
- Contains 7.5% NaCl - inhibits nearly all other groups of bacteria and some speceies of staphyloccci
- Contains mannitol and organisms which produce acid fro manitol will produce an acid change in the medium
- Inoculate a batriera spot about the size of a dime. Incubate 18-24 h at 37C
- Differential and Selective
What is the Motility Test?
- Purpose is to determine if an organism is motile or non-motile under the growth conditions of the test
- Contains 0.5% agar and is semisolid - allow movement
- Inoculate with a needle by stabbing directly down the center of the tube down to as close to the bottom as possible, and draw the needle out in the same direction
- incubate at room temperature for 24-48h
- Turbidity from the stab = Positive
- Turbid only in stab = Negative
- General purpose differential
Due to lack of oxygen at the bottom of tube, organisms that are stricktly aerobic and motile may appear non-motile (false negative)
What is an Oxidase test?
- Identifies bacteria which produce the enzyme cytochrome oxidase and are able to oxidize a dye tetramethyl p-phenylenediamine to indophenol (colored)
- Differentiate Enterobacteriaceae from:
- Neisseria spp. and Morazella catarrhalis are oxidase positive
- Psuedomonas spp(except Burkholeria cepacia)
- Aeromonas
- Use a cotton swab to collect a visible amount of growth from Blood agar plate
- Add 2 drops oxidase
- Purple (within 15 seconds) = Positive
- Purple (1-2 minutes) = False positive (except Pasteurella/Mannheimia spp)
- No change = Negative
Dyes in MacConkey can lead to false negative due to excessive acid
Tetramethyl p-phenylenediamine is unstable - will autoxidize - do not use if deep blue
What is Phenol Red Broth with Mannitol?
- Distinguish between Bacillus anthracis or Bacillus cereus (negative) from Bacillus subtilis (positive)
- Contains pancreatic diget of casein, sodium chloride, a carbohydrate, and phenol red
- Contain Durham tubes to visualize gas production
- Innoculate with isolated colony from a young culture
- Inncubate 35-37C for 3-5 days
- Yellow = Positive
- Red = Negative
- Gas = gas fermentation reations
- General purpose differential
What is Phenol Red Broth with Trehalose?
- Ability to ferment Trehalose to produce acide can distinguish between Streptococcus suis (positive) and Streptococcu zooepidemicus (negative)
- Has a sugar-free basal medium, phenol red, and 1% trehalose
- Inoculate with an isolate from a yong colony and incubate at 35-37C for 3-5 days
- Yellow = Positive
- Red = Negative
- General purpose differential
What is a Spore Stain?
- 2 major spore forming genera Bacillus and Clostridium - cause botulism, gangrene, tetanus, anthrax, etc
- Malachite green is weak binding to cell/spore wall
- Use heat to help lock in the stain to the spore wall
How do you perform a Spore Stain?
- Prepare smear, air dry, and heat fix by passing through flame 3x
- Cover smear with Malachite green and pass 20x 4 inches above flame
- should steam for ~30 seconds but not dry out
- Let cool
- Rinse with tap water
- Flood slide with safranin for 30 seconds
- Rinse, Dry, examine under oil immersion
What are Triple Sugar Iron (TSI) Agar Slants?*
- Indicates the ability of an organism to ferment lactose, sucrose, and dextrose with formation of acid, possible gas, and also the ability to produce hydrogen sulfide
- Inoculate slant with loopful of culture
- inoculate the butt by stabbing with needle
- Incubate at 37c for 24h (may take longer)
- Reactions:
- No change = No change in color
- Alkaline (K) = more Pink/Red
- Acid (A) = Yellow
- Hydrogen Sulfide (H2S = Black
- Gas (G) = gas bubbles in butt
What are the interpretations of TSI slants?*
- NC/NC: either does not act on any of the carbs, or is an O-F oxidizer and requires oxygen
- K/NC: Organism has acted on the proteinaceous compounds. O-F should be run as above
- K/A: Organism is a Fermenter and produces acid from only glucose not from lactose or sucrose. Also producing alkaline products
- K/A + H2S: Organism is a Fermenter and produces acid from only glucose not from lactose or sucrose. Also producing alkaline products. H2S is produced from sodium tiosulfate. If it obscures results run an O-F
- K/NC + H2S: Organism has acted on the proteinaceous compounds. O-F should be run. H2S is produced from sodium tiosulfate. If it obscures results run an O-F
- A/A: Bacteria are known to be fermenters in O-F medium, Ferment lactose and/or sucrose and glucose
- A/A + H2S : Bacteria are known to be fermenters in O-F medium, Ferment lactose and/or sucrose and glucose and produce H2S.
- Erysipelothorix rhusiopathiae small black line
- Proteus vulgaris large amount
- A/A + G: Bacteria are known to be fermenters in O-F medium, Ferment lactose and/or sucrose and glucose ad produces gas
What are Urea Agar Slants?
- Deterimes if an organism has the ability to produce the enzyme urease which will split urea into two molecules of ammonia
- Inoculate slant heavily with growth from any medium Incubate 37C for 24-48h
- Pink = ammonia = Positive
- Yellow = Negatie
What is Rappaport-Vassiliadis (RV) Enrichment Broth?
- Used for the isolation and cultivation of Salmonella species from food, clinical specimens, and environmental samples
- Enrichment results in a higher tolerance to malachite green by Salmonella relative to other members of Enterobaceriaceae