microbio
MODULE 6:
glucose fermentation - turns yellow in presence of acid (positive test)
gas must take up at least 10% of the durham tube to be considered a positive result
nitrate reduction test -
denitrification: NO3 to N2 look for a large bubble in the durham tube
no need to continue the protocol is this occurs
to test for NO3 to No2 add drops of each of the two nitrite reagents (sulfanilamide and N-ethylenediamine)
pink or red color = presence of nitrite
adding zinc to tube
pink or red color = negative reaction for nitrate reduction by the organism
catalase tests add hydrogen peroxide to the microbe and bubbles indicate positive reaction
strict aerobes and facultative anaerobes would be expected to possess the enzyme catalase
MODULE 7: MICROBES IN FOOD AND WATER
indicator organism - used as a measure of fecal contamination to assess water quality, 4 properties:
advantage - easy to test for
disadvantage - doesn’t truly tell you if there is a pathogen there or not, just tells you if there is likely to be a pathogen
only natural environment of the microbe should be in association with feces and it should be always present
should not grow in nature outside of its natural environment
bacterium should survive longer than the most viable pathogen, but not so long so that historical events are detected
should be easy to detect
enterobacteriaceae - family of gram negative Proteobacteria that comprises a very large group of morphologically and physiologically similar bacteria that have a wide range of positive and negative impacts on human, animal, and plant health
All enterics are
gram negative rods
chemoorganoheterotroph and true facultative anaerobes
can all ferment glucose and often other sugars and are also capable of aerobic respiration of various organic compounds
catalase positive
oxidase negative
most are motile via flagella
coliforms - closest to fulfilling criteria of indicator organisms and are used as a measure of biological contamination of water
gram negative rods that ferment lactose rapidly with the production of gas
LGLB mildly selective test
BGLC coliform confirming tubes
EC confirming fecal coliforms
grown at higher temp (44.5)
KIA - used only for gram negative bacteria, mainly enterics and pseudomonas
purpose is to test fermentation of a major sugar (lactose) and minor sugar (glucose) and the production of hydrogen sulfide
H2S reacts with iron in medium to produce ferrous sulfide (black precipitate)
protocol - medium is inoculated by stabbing down center to bottom then streaking surface of the slant
yellow color at bottom indicates isolate is capable of minor sugar fermentation
red at bottom indicates glucose
yellow at top indicates major sugar fermentation
black precipitate indicates production of hydrogen sulfide
indole production - examine tube for growth and if growth occurs then continue with protocol
add few drops of Kovac’s reagent
development of a red ring at the top of the medium is a positive indication of indole production
methyl red - purpose is to identify the end products from glucose fermentation
tests for the relative pH attained from glucose fermentation in a standard medium
organism is positve for MR or VP not both
if it doesn’t stay red then it’s probably VP
red - positive
yellow - negative
orange - equivocal reaction
simmons citrate - tests for use of citrate as a carbon and energy source
bright blue indicates positive test
test is known for giving false results
EMB - inhibits growth of gram positive bacteria typically used for gram negative only
colonies of lactose fermenting bacteria will show a relatively dark color while lactose non fermenters will produce white or pale colonies
coli-type - colonies appear dark, almost black when viewed directly and have green metallic sheen if observed by light
aerogenes-type - less dark colonies, light colored rim surrounds dark center (fisheye colony)
Phenylalanine deaminase - test for deamination of the amino acid phenylalanine
streak across the top of the slant only
add FeCl3
dark green color on slant indicates positive, may take several minutes
MIO
motility - cloudiness in medium
ornithine decarboxylation - look at lower three quarters (anaerobic region), yellow color is negative reaction which is due to acid production from glucose fermentation
gray, blue color is positive indicating formation of a highly alkaline product, over neutralizing the acid produced from glucose fermentation
indole production - add one half dropper of kovacs reagent, red ring indicates production of indole from breakdown of tryptophan
Lysine decarboxylase
overlay the growth with mineral oil and incubate
yellow color = negative
gray, blue = positive
Decarboxylase control - similar as lysine
lactose fermentation
purple to yellow color indicates lactose was fermented to acid
colilert test - predicts the most probable number of coliforms and fecal coliforms in water samples
the likelihood of finding a positive result in a given pouch volume is calculated and the total number of positive results are tallied
pouches testing positive are tallied and formula is applied to determine the most probable number of bacteria in original sample
results:
if coliforms are present ONPG is metabolized results in yellow color
MUG will indicate presence of fecal coliforms
MPN:
use middle dilution factor of the three that you use
MODULE 8:
first to grow are strains of enterics that are identified as enterobacter and klebsiella
then lactic acid bacteria predominate as pH drops
after Leuconostoc peaks then highly-acid tolerant Lactobacillus takes over
lactic acid bacteria - gram positive rods and cocci
lactobacillus, streptococcus, enterococcus, lactococcus, leuconostoc
produce lactic acid as primary end product of glucose fermentation
heterolactic fermenters produce equal amounts of lactic acid, ethanol, and carbon dioxide
generally aeroteolerant anaeorbes
also obligate fermenters that are incapable of aerobic or anaerobic respiration
heme-containing compounds associated with respiration and H2O2 breakdown are absent in lactic acid bacteria
fastidious organisms - requires media that contains many growth factors
resistance of these to azide is exploited by the inclusion of sodium azide in the media
MODULE 9:
antimicrobial agent - chemical that kills or inhibits the growth of microorganisms
cidal - agents that kills organisms
static - don’t kill organisms may be bacteriostatic or fungistatic\
antiseptics - microbicidal agents harmless enough to be applied to skin and mucous membrane but not taken internally
iodine, alcohols, detergents
disinfectants - could kill microbes but not necessarily their spores
used on tables, floor, utensils not skin
preservatives - static agents used to inhibit the growth of microbes in foods
calcium propionate, sodium benzoate, formaldehyde, sulfur dioxide
antibiotics - kill or inhibit other agents
gram positive - must pass through thick layer of peptidoglycan
gram negative - pass through porins in outer membrane
types:
ampicillin - affect peptidoglycan synthesis
bacitracin - affect peptidoglycan synthesis
ciprofloxacin - affects DNA replication
kanamycin - affects proteins synthesis
tetracycline - affects protein synthesis
zones of inhibition calculation
supplied concentration (mg/mL) x (1/1000uL) x (1000ug/1mg) x con in uL
used to confirm on table if it the calculation is the same as the value
lower limit of detection - take the lower limit 30 x lower dilution (10-1)
upper limit of detection - take upper limit 300 x highest dilution (10-4)
fecal coliforms grown at 37 instead of 45 what happens is the non fecal coliforms are not killed
supposed to incubate at 44.5
false positive for fecal coliforms
pour plate dilution - 1 mL
spread plate dilution - 100 microliter
count an extra 10 for the dilution factor