LAB PRACTICAL 1 Flashcards
Heat fixation
bacterial smear will wash away during staining procedure, so the bacteria proteins are coagulated to the glass surface by heat fixation
B. cereus
Bacillus cereus -gram positive -spore forming -rod shaped -facultative anaerobe FERMENTATION: dextrose- produces acid sucrose- produces acid **DOES NOT FERMENT LACTOSE** AGAR SLANT GROWTH: -abundant, opaque, white waxy growth
S. aureus
Staphylococcus aureus -gram positive -facultative anaerobe -little cluster of small cells FERMENTATION: lactose- produces acid dextrose- produces acid sucrose- produces acid AGAR SLANT GROWTH: -abundant, opaque, golden growth
simple stain
when a bacterial smear is stained with a single reagent
methylene blue or crystal violet
Methylene blue
good for seeing morphology
STAINS ALL BACTERIAL TYPES
negative staining
requires the use of an acidic stain like nigrosin that is negatively charged so it stains the background around the cells but bacterial cell walls in gram positive are negatively charged and gram negative cell membranes are negatively charged so he stain does not enter the cell.
THESE STAINS ARE USED FOR SEEING CELL SIZE AND MORPHOLOGY
Procedure:
-place a drop of nigrosin at one end, place loop of culture into nigrosin drop and slide the mixed drop along the slide with another glass slide
E. coli
Escherichia coli -gram negative -facultative anaerobe -rod shaped -white on tryptic agar slant FERMENTATION: lactose- gives off acid and gas dextrose- gives off acid and gas sucrose- (variably reacts) AGAR SLANT GROWTH: -white, moist, glistening growth
M. luteus
Micrococcus luteus -gram positive -small clusters of cells -strict aerobe -yellow on agar slant FERMENTATION: **NO FERMENTATION of lactose, dextrose, or sucrose** AGAR SLANT GROWTH: -soft, smooth, yellow growth
Differential staining
requires the use of at least four chemical reagents that are applied in sequences
- Primary stain- crystal violet
- Mordant- Gram’s iodine (increases cell’s affinity for the primary stain)
- Decolorizing agent- 95% ethyl alcohol (washes out unbound crystal violet-iodine complex from gram-negative cells)
- Counterstain- stains gram-negative cells pink
Acid-fast stain
better technique of viewing Mycobacterium, which can be pathogenic to humans
1. Primary Stain- carbol fuchsin (dark red stain that is held by the mycobacterium)
2. Decolorizing agent - acid-alcohol (3%HCl and 95% ethanol)
3. Counterstain - methylene blue (stain held by other cells that do not retain the carbol fuchsin)
PROCEDURE: apply carbol fuchsin to slide with mycobacterium and put the slide over boiling water on a hot plate for 5 minutes (the heat helps the carbol fuschin penetrate the cell wall)
M. smegmatis
mycobacterium smegmatis
- small clusters
- rod shaped
- strict aerobes
- have a waxy lipoidal cell wall that doesn’t hold stains like methylene blue or crystal violet
- found clustered together
- these organisms are considered “acid fast”
Spore Stain
- Primary stain- malachite green (must be heated first and then both the vegetative cell and the spore will appear green)
- Decolorizing agent- water (malachite green doesn’t bind as well to vegetative cell, so water washes the green off of the vegetative cell)
- Counterstain- safranin (this stain is held by the vegetative cell)
C. sporogenes
Clostridium sporogenes
- species of gram positive bacteria
- can form spores
- rod like and appear in chains
- strict anaerobe
Streak-plate method
inoculate a Trypticase soy agar plate and drag the loop back and forth over Area 1. flame the loop and turn the agar plate slightly (90 degrees) and drag culture from Area 1 into Area 2 but do not let loop go back into Area 1. Repeat this procedure until four sections of plate are inoculated
S. marcescens
Serratia marcescens
- rod shaped
- gram negative
- facultative anaerobe
- red on agar slants
P. aeruginosa
Pseudomonas aeruginosa
- strict aerobe
- rod shaped
- gram negative
- produces enzyme gelatinase and liquefies gelatin agar tubes
- *NO FERMENTATION**
- white with greenish tint on agar plate
Selective media
inhibit the growth of one type of bacteria while permitting growth of another type
Examples:
Phenylethyl alcohol agar
Phenylethyl alcohol agar
inhibits gram negative and allows for gram positive growth
Differential media
slightly selective but allows for differentiation of closely related organisms based on biochemical makeup of organisms (whether they break down certain nutrients or not) Examples: Mannitol Salt agar MacConkey agar Eosin-methylene blue agar
MacConkey agar
DIFFERENTIAL MEDIA
- inhibitory effects of the crystal violet in this agar on gram positive organisms allow for the isolation of gram negative organisms
- this agar has lactose, so organisms that can ferment lactose turn red on this agar and pink can sometimes form around the red growth (pink growth for E.coli)
- organisms that don’t ferment lactose appear lightly tan or transparent
E. aerogenes
Enterobacter aerogenes -rod shaped -gram negative -facultative anaerobe FERMENTATION: lactose, dextrose, and sucrose all cause acid and gas creation
S. typhimurium
Salmonella typhimurium -gram negative -rod shaped -facultative anaerobe FERMENTATION: dextrose and sucrose (NO LACTOSE)
Mannitol salt agar
DIFFERENTIAL MEDIA
-this media is selective because only staphylococcus organisms can grow, but is differential because depending if a staphylococcus species can ferment mannitol or not (if mannitol is fermented, growth appears yellow)
Eosin-methylene blue agar
DIFFERENTIAL MEDIA
- partially selective for gram-negative bacteria and differentiate (isolate and show differences) of enteric bacteria because this agar has lactose.
- E.coli turns metallic green on this agar and E. aerogenes turns a thick mucous pink
