Micro Lab Final Flashcards
Set up Kholer illumination
refer to lab manual 2-9
Smear prep from broth culture
manual 3-6
gram stain
3-7
streak plate from broth
4-3
cell morph (terms and table prep for g stain)
4-13
colony colour
g rxn (-/+)
cell shapes
cell size (um)
cell grping
mic clean up
discard slides in beaker
remove oil from 100x lens w paper
10x lens and aperature
turn off light
set up serial dilution
manual 5-5
spread plate
manual 5-6
why is a sample turbid
cloudiness bc scattered light (more cells = more scatter = greater turbitity)
how do we meas turbidity/scattered light
spectrophotometer can meas dif bw amnt of light that enters sample and amnt of unscattered light that leaves sample
unit = optical density (OD)
what is optical density
directly proportional to number of cells in sample
as turb increase, so does OD
allows us to see total number of cells
why are OD meas’s better than plate count method
same broth can be checked many times and can track rate cells numbers increase
what is a standard curve
scatter plot that relates OD (y axis) to cells per mL (x axis)
to est standard curve, need to count # of cells in sample you’re taking OD reading from
get cells #’s by plate count method
what are the lim’s of OD to cell #’s relationship
- direct rel bw OD and # of cells/mL breaks down at high turb (rel strongest bw 10^7-9)
- a/t that can affect cell size/shape/grpings will change standard curve (make new curve is change org, growth med, incub condition)
- some bact form clumps/long chains as rep/divide and some grow in films (vortex before sampling to avoid)
- dif media have dif baseline optical densities dep on colour/pigmentation
what is imp to know ab standard curve
stand curve does not provide any info ab how quickly a pop is growing
what is the cell density meter set to
600 nm for bact and yeast (not for mammalian cells)
why should the red set be redone every 15 min in density meter
avoid drift (use NB and press R)
when looking at series of OD readings, how do you check if they’re valid
should drop by half
what are coliforms
indicator organisms
from fam enterobacteriaceae that serves as index for potential contam by entero pathogens
safer, cheaper, easier faster
what do bacteriological evaluations of water meas
specific subpops of bact rather than testing for every potential pathogens individually
quality of water det’d by kinds of bact present not how many
what do water quality analytical procedures and purification/treatment methods rely on
vol, source, initial quality of water
what is the total coliform (TC)
grp of bact includes many dif genus and species
coliforms found in many env including intestinal flora (imp habitat bc many coliforms are thermotolerant and passed through fecal wastes)
why are coliforms used as indicators for potential contam
- natural habitats are imp sources of water contam
- capacity to survive fresh and marine water is similar to that of entero-pathogens
- density usu correlates w degree of water contam
- do not reprod in water (as long as water is not nutrient laden)
how do we distinguish coliforms
facultative aerobic
g neg
rod
non-spore forming
non motile or motile by peritrichous flagella
ferment lactose w prod of gas w in 48h at 35C
what is dif ab thermotolerant coliforms
usu of fecal origin
prod acid (alongside gas) w in 24 h at 44C
if get thermotol fecal coliforms, indicates fecal contam
what is the most common fecal coliform
Escherichia coli
what is imp to remember ab testing for coliform contam
most coliforms are not pathogenic but presence in water indicates the likely presence of pathogens
two techniques to test for total coliforms and/or thermotolerant (fecal coliforms)
membrane filtration method
most probable number of coliforms technique
what is selective media
permit or enhance growth of desired org while suppressing/inhibiting growth of another
what is differential media
support growth of dif orgs but allow orgs to be distinguished phenotypically based on their biochem
how are g neg selected for medium in lab 7
bile salts, detergents, amphipathic dye salts
what do bile salts do and how do enteric bact resist
bile salts destroy lipid bilayer of cell membrane
enteric bact resistant to them bc have crosslinked matrix which acts as exclusion barrier to phobic mols and amphi mols like detergents and bile salts and neg charge mols
what also select for g neg
BG, basic fuschin, rosolic acid, aniline blue which are ph indicators
cant penetrate LPS of g neg but can stop growth of g pos
how are coliforms differentiated from other g neg orgs
based on ability to ferment the sugar lactose w prod of gas
when does fermentation occur
when bact/yeast import and break down a carb (sugar) from surrounding medium/env to release E w production of alcohol and/or acid and CO2
ETC not involved and e A usu pyruvate
what is resp
uses ETC w O or a dif mol as final e A
how to detect non soluble gases that may be produced during fermentation
use broth medium w durham tube
tube collects gas prod’d by bact during incubation
how to detect acids prod’d during fermentation
add ph indicator dye to growth medium in addition to specific carb
what is M-FC agar selective for
thermotolerant g neg bact based on ability to grow in presence of high incubation temps, bile salts, rosolic acid and aniline blue
differential for lactose fermenting abilities on basis of ph of major end prods
what colour do mixed acid lactose fermentors (e coli) produce
dark blue colonies bc drop in ph
what colour do butadienol lactose fermentors (enterobacter) appear
blue to blue grey (butad raises ph)
what colour do lact fermentors appear
pink-reddish blue grey
salmonella and shigella
issues of tsa plates
obs of colonies are often difficult due to crowding
no info ab hazardous bact vs harmless bact
max vol plated = 100 uL
how does the membrane filter (MF) work
water passes thru sterile mem filt w pore size 0.45 um and only things smaller pass
filter then placed on sel and dif med for thermotolerant fecal coliforms (44.5C)
MF advantages
- range of vols can be processed (can be large, up to 10L)
2.water sol impurities that might interfere w indicator org growth pass through small pores and removed from growth medium - provides precise quantitative enumeration
- usu only need single incubation step (fast and simple)
- colonies are sep’d on filter, makes downstream isolation easier
MF disadvantages
- cant be used to test waters w high turbidity w out pre filter bc filter will clog
- scoring of colonies (differentiation) can be difficult if sample contains large numbers of non coliform bact and excessive crowding occurs in filter
- solid particles and chems may absorb to filter and interfere w growth coliforms
calc fecal coliforms/100mL
fecal coliform colonies x 1/dil
rules for M-FC
- lim 20-60 fecal coliforms (blue)
- <200 total col
put est if more than 200 or if no col present
what is MPN of coliforms
indirect quant analysis
pos test indicated by growth and gas
advantages of MPN
- flexible vols can be processed (<10mL)
- all kinds of water samples can be used
- stressed and injured coliforms can be recovered
- test is easy to set up and results easy to interpret
- precision and sensitivity can be increased w larger vol, many diltuions or greater number of replicate tubes
- media is cheap
MPN disadvantages
- only semi quantitative
- precision is lower and relies on number of tubes used in test
- presumptive phase takes 48h + 24 (fec)/48h(tot) subculture confirmation
- if isolation req’d, need more steps on solid media
- coliforms can be suppressed or overgrown
- water sol impurities can affect coliform growth
- hard to process large vol
what is the presumptive coliform step
enrichment step
medium contains peptones and lactose
g pos and neg can grow but s/t get false pos (would need selective media to confirm)
must produce gas and grow
what is the confirmed (total) coliform test
only use pos from presump test
use brilliant green lactose broth (has peptones, sel dyes, lactose, bile salts)
need growth and gas
what is the fecal (thermotolerant) coliform test
from presump tests that were pos
medium selects for thermotol coliforms w 44.5C inc temp
growth and gas
aka EC broth tubes
what are the recreational water quality standards
health can - <500 TC/100mL
<200 fecal coliforms/100mL
<35 enterococci/100mL
US env protection agency <200 fecal coliforms/100ml
WHO <100 e coli/100 ml
what are drinking water quality standards
no coliform pres/detectable
what are enteric bact
oxidase-neg, catalase pos, prod acid from glucose, reduce nitrate to nitrite
many = chemoorganotrophs
hard to ID bc all g neg rods that are beige cols, round smooth
how do enterobact break down sugars
embden meyerhoff pathway/glycolysis
happens during:
aerobic resp (e A = O; etc), anaer resp (NO3, CO2 etc; etc), ferm (pyruvate, not etc)
what are the dif categories of ferm and prods generated useful for
ID process of orgs in ent fam
key pts when looking at microbial fermentations
NADH ox’d to nad+
O2 not req’d
term e A usu pyruvate and no etc
what are mixed acid fermentors
prod ethanol and stable acids
what are butanediol fermentors
prod butanediol, ethanol, co2, some lactic acid and formic acid
what is the phenol red broth test
general medium, not sel
have durham tube
dye = yellow at low ph; bright pink at high ph
if bact can ferment test carb, acidic end prods lower ph (yellow) and get growth
if no ferm, no acids, still get growth but neg, high ph (orange/red)
what is MRVP broth test
to dif enterobact based on major ferm end prods when supplied w glucose
MR - red at low ph, yellow at high
mixed acid ferm’s prod stable acid end prods and lower ph
VP - detects for presence of acetoin (precur of but)
brown-red = pos
brown green-yellow if neg
citrate slant
sel for bact that can use citrate as sole c source
bromothymol blue dye = green at low ph/blue at high ph (blue if pos)
urea broth
differential for bact that are rapid urease pos (hydrolyze urea fast)
pos = high ph = neon pink
neg = pale orange/pink
TTC motility
use triphenyl tetrazolium chloride semi solid agar medium
red colour where cells have grown (all enterobact can grow) but will see red beyond stab line if mobile
SIM
- tests for sulfur reduction (get black precipitate)
- tests for tryptophan deamination (bact that prod tryptophanase can deaminate aa tryptophan and get indole); use kovac’s reagent
- motility (look for cream colour growth beyond stab line)
which media are dif and sel
an blue, bril gr, carmen red, bas fusch
which media is sel
bile salts
which media is dif
phenol red
Ingredient that allows the media to be differential for lactose fermentation
rosilci acid
Ingredients that inhibit gram positive bacteria:
aniline blue, bile salts
pos/neg phen red
yellow/red
pos/neg mr
red/yellow-orange
pos/neg vp
brown red/brown green
pos/neg citrate
blue/green
pos/neg urea
bright pink/pale orange
tryptophan deamination pos/neg
reagent layer bright pink/yellow-orange
