week 1 objectives Flashcards
why do some cells stain gram + and gram =
gram + bacteria has lesser amounts of lipid contents in its cell wall
gram = bacteria cell walls have more lipid and don’t get dehyrdated during the alcohol phase and loses the stain and takes up the counter stain
gram stain reagents
primary stain - crystal violet
mordant - grams iodine
decolorizer- acetone / ethyl alcohol
counterstain - safranin
how would you prepare a swab specimen
the smear will be made following plates and any broths.
made by dabbing and rolling both sides of the swab ovr a quarter size towards middle of slide.
then heat dry, then stain, and dry once more
how would you prepare a fluid specimen
over a certain amount will be spun down and the supernatant will be gently removed leaving about 1 cc of specimen that will be resuspended. then placing one drop on glass slide, and spread out then heat, stain, dry again etc.
bacteriocidal
kills the organism
bacteriostatic
retards growth of the organism
identify specific precautions that can be used to control infectious aerosols
- when streaking plates don’t allow loops to bounce off sides of plate
- cool inoculating loops/needles by holding in air 10-15 seconds, or use 2 loop needles and alternate
- don’t plunge hot loops/needles into media to cool
procedure for culture spill/drop
first spray the area with hospital approved disinfectant, then cover the area with paper towels that are saturated with disinfectant. let this stand for 15 minutes then using gloves and new paper towels scoop up all of contaminated material and deposit in biohazard trash can.
why can gram + organisms may stain gram gram = when old colonies are picked for gram stain
because of the age of the organisms cell walls have lost their integrity
what is the purpose of streaking
isolate colonies of bacteria to try and see potential pathogens
describe 3-4 zone streaking technique
initial inoculum is placed in the first quadrant, then with disposable loop (unless sterile site) the initial inoculum is spread, then spread right side of 1st quadrant into a 2nd quadrant clock wise around plate. obtaining new loop, the third streak will run over into fourth quadrant branching off prior streak
regarding thioglycolate media, describe zones of growth for 1 obligate aerobes 2 obligate anaerobes 3 facultative organisms 4 microaerophilic organisms
1- zone of growth is shown right below surface of agar
2- zone of growth is in the butt of the tube
3- zone of growth is the entire tube
4-zone of growth is right below obligate aerobes
obligate aerobes
require atmospheric oxygen and posses enzymes to break down toxic end products of atmospheric oxygen
obligate anaerobes
atmospheric oxygen is toxic. these organisms lack enzymes to break down toxic end products of atmospheric oxygen
facultative organisms
can metabolize aerobically or anaerobically
microaerophilic organisms
grow best in atmospheres of reduced oxygen tension
what is the preferred blood for making blood agar plates and why is it preferred?
sheep blood is preferred because streptococci may show erratic or different hemolytic reactions
human blood may contain nonspecific inhibitors of microbial growth (antibodies, complement, antibiotics) blood from BB may have excess citrate that may inhibit some gram + organisms
what makes chocolate agar more enriched than blood agar if the two media are made with the same ingredients
chocolate agar is added to the plate while still hot so it chocolatizes the blood coagulating proteins, releasing NAD from erythrocytes and hemin
know the two growth factors provided by chocolate agar that allow H. influenzae to grow
hemin and NAD
know two organisms that will allow grow on chocolate agar but will not grow on blood agar
Neisseria gonorrhea
Haemophilus influenzae
what does PEA stand for and how/why is it used?
phenylethyl alcohol agar
selective for gram + (staph/strep) inhibits growth of gram = by inhibiting synthesis of DNA for only so long before they start growing
why should PEA not be used to determine hemolytic reactions
because atypical reactions may occur
what organisms is Mcconkey selective for and how it differentiates these organisms
gram = organisms and lactose fermenters
gram = will grow, gram + no growth
lactose fermenters are pink
NLF are clear
be able to list 5 gram negative bacteria that will not grow on Macconkey
Actinobacillus pasteurella moraxella Pseudomonas mallei flavobacterium
know why indole or oxidase tests should not be performed from colonies growing on Macconkey agar
because growth on Mac inhibits/suppresses enzymatic activity
ex. Aeromonas growth on Mac may show oxidase =
be able to list two organisms other than gram = bacteria that will grow on Mac agar
yeast
know what HEA stands for. know what organisms it is selective for and how it differentiates these organisms
Hektoen Enteric Agar
selective for GNR (shigella/salmonella)
pathogens are clear green or black
nonpathogens are orange-salmon colored
know what two organisms modified thayer martin is selective for
Neisseria gonorrhea and Neisseria meningitidis
list the three antibiotics additives in MTM and modes of action
vancomycin- inhibits gram + bacteria by interferring with cell wall synthesis
colistin- disrupts permeability of the cell wall of gram = bacteria
nystatin- inhibits yeast by altering permeability of the cell wall
trimethaprom- prevents proteus from swarming
know what genus laked blood agar with kanamycin and vancomycin is selective for
bacteroides
know why gram negative broth is used and what the ideal subculture time is
stools- shigella / salmonella
6-8 hours
differential media
displays a visual difference between colonies growing on media
selective
a medium with substances that allow preferential growth of one group of organisms over another
reagents of AFB
carbfuschin - primary stain - five minutes
acid alcohol - decolorizer- 2 minutes
methylene blue - counter stain - 3 minutes
what is the difference between Ziel-Neilsen and Kinyoung method?
kinyoun doesnt use heat fixation
what organisms will stain acid fast?
mycobacteria and nocardia
how are acid fast differentiated from nonacid fast bacteria using the kinyoun method?
AFB will be fuschia/hot pink and all other will be purple/blue
describe principle of AnaeroPouch systemfor anerobe jars
a single-use disposable anaerobic gas generating system designed to produce conditions suitable for cultivation of anaerobic microorganisms w/o use of water and/or catalyst. Atmospheric O2 is rapidly absorbed with simultaneous generation of CO2
specimen handling/transport times/ specimen collection storage etc
plate urines/sputums/fecals within 2 hours
wound cultures within 1 hour
CSF, BF, sterile sites STAT
gram stains should be done and reported out within 1 hour
urines get refrigerated
sputum, fecals, wounds, CSF- room temp
all must be in sterile contains excpt sputum and fecals can just be in clean, uncontaminated containers
why are sputum specimens screen with a gram stain?
they are screened to determine quality of specimen
what specific sputum specimens can be rejected based upon the gram stain result?
expectorated sputums can be rejected if there are is an abundance of epithelial cells contained
when is the best time to collect sputum for AFB cultures
first morning
anaerobic jar
anaero-pack rectangular jars, sashe, indicator strips, hospital approved disinfectant, 4X4 gauze
sashe goes in B compartment
indicator strips in C
up to 12 plates in AGAR SIDE UP
close and place in O2
campy pouch
baggie- add plate (and buddy if not 2 plates)- sashe (little blue ones) - date - and close put in CAMP incubator
anaerobic pouch
baggie add 1 plate at a time, indicator strip, sashe, date , close and put in O2 incubator
aseptic technique
the method of working that prevents the spread of microorganisms
what does C. neoformans in india ink look like?
cryptococci are spherical (sometimes oval) yeast forms which range from 4 micrometers to 20 micrometers.
has a polysaccharide capsule that remains unstained “clear halo”
procedure for mislabeled specimens
are rejected by calling sender, notifying them the order will be canceled and other specimen will need to be resent
exception: irreplaceable specimens the responsible party must come and identify specimen and label themselves
what are the consequences of delayed transport in regards to anaerobes, overgrowth of normal flora, and culture validity
anaerobes will die
overgrowth of normal flora will mask any potential pathogens
culture validity decreases
know for what tests/specimens refrigeration is acceptable storage temperature
urines, specimens for AFB, fungal testing on respiratory specimens, stool for C.diff if testing cannot take place within 24 hours
MRSA/VRE when testing cannot be done within 24 hours
know what CSF tube is routinely used for culture and gram stains unless stated otherwise by the physician
TSB- tryptocase soy broth, promotes fastidious organism growth, considered enrichment broth
why do you want to avoid cross contamination with specimens for MRS/VRE/C. diff
because with these tests you are dealing with DNA/RNA which are so easy to pick up and transfer causing erroneous test results
