w10 micro Flashcards
what are media QC procedures?
- documentation of lot number
- prep dates
- rotate media storage so old media used first
- discard outdated media
- media performance testing (pH, sterility, viable quality of organisms)
sterility checks
- not usually done with purchased media
- incubate plates at 35 degrees C-colony growth indicates contamination.
functional checks
- determine media performing properly.
- each lot number tests, documented.
- inoculated to culture medium and incubated (specific qc strains selected for behaviour in certain media)
what do functional checks test for?
- if certain organisms inhibited
- nutrients required for growth of fastidious organisms present
- bacteria appear as the should
- expected colour changes occur
general definition of C&S
culture- identify bacteria, sensitivity- susceptibility to antibiotics
what technique is used for collecting C&S specimens?
aseptic technique - ensure specimens not contaminated and potential pathogens in specimen is present. *collect before antibiotics.
what to do if delay in transport for C&S
refrigerate if they cannot be transported promptly. It will prevent growth of bacteria but not kill them
amies media swab (pink or red lid)
throat
wound
urogenital
charcoal media swab (black lid)
urogenital
blood culture bottles (anearobic and aerobic)
for blood
boric acid tubes (for collecting C&S)
urine
pre-reduced anaerobic sterilized PRAS transport media
tissue and bone
how to treat CSF and why?
treat as stat -***should never be refrigerated, Meningitis bacteria may be killed by the cold
How to prepare sputum for C&S?
-good specimen contains mucus- us a mucolytic agent for 15-20 and then mix with vortex to use
How to prepare tissue for C&S
-ground up to release microbes
How to prepare fluids for C&S?
-CSF, Pleural peritoneal fluid require concentration with a centrifuge or serofuge
microcentrifuge for fluids
- rapid concentration of small volumes.
- Specimens transferred into small conical sterile plastic tubes with snap lids
- concentrated in a few minutes
filters
- can be used to prepare sterile fluids or for Culture media
- once fluid through, filter removed with sterile forceps and placed on surface of appropriate culture media
appropriate culture media for different body cites
- specimens grouped according to body sites and microorganisms encountered in those areas.
- varies from lab to lab depending on patient populations and specialized nature of some laboratories
media to inoculate throat swab?
-BAP with taxos A disc, incubated anaerobic (anO2)
Add a staph streak if patient older than 5
Media to inoculate eye swab?
-Direct smear BAP with staph. streak Chocolate CO2 MTM CO2 BHI broth
plural fluid: cytofuge or centrifuge to concentrate
- Direct smear
- BAP
- Chocolate CO2
- Brucella Blood Agar anO2
- PEA anO2
- RCM
how is the method of inoculating specimen determined?
it is determined by type of specimen- swab, liquid, semisolid
how are swabs inoculated
used to make smears and inoculate (crosshatch and circle) all required media
how are liquid specimens inoculated
- sterile swab
- sterile pasturer pipette
- sterile loop
how are semisolid specimens inoculated
-sterile swab- stool, sputum
Order of Inoculating media
- inoculate nonselective media first and most selective media last- prevents inhibitory agents from being transferred from a selective media to a nonselective media
1. Direct smear
2. solid plate media with non-selective inoculation ( BAP, CHOC, MAC) then selective second.
3. liquid media last, swab left there.
what is a direct smear
-specimen smeared on glass and is stained for examination for presence of bacteria and neutrophils
–monolayer of cells in clinical exudate
made from swabs or pasteur pipette
how to make direct smear
- clean/ sterile slide
- roll swab over labelled slide- all sides of swab make contact with side
- lots of exudate on swab-spread over larger area
- should not be thick layer
- direct smear before specimen is planted
what to do if two swabs submitted from site
-only smear and inoculate one, the other is saved
amount of bacteria in culture
- graded light to heavy
- clinical significance determine the significance of bacteria isolated – amount of growth determined by number of bacteria in specimen
stool specimens
-plated with increased amount of inoculum on selective media- gives better chance of isolating specific pathogens– not graded for amount of growth only presence
what does streaking accomplish?
- spreads- isolated colonies
- pure culture required for identification of all bacteria
- allows grading of amount ** must be standardized
why must streaking must be standardized?
-amount of growth related to number of bacteria not how plate was streaked
variables of streaking technique that affect results
- angle of loop: wide=more bacteria, narrow=less bacteria
- speed: faster= less bacteria dragged along
- changing stick/sterilization: new= reduce carry-over from previous set of streaks
why use sticks for streaking?
- light touch-dig into agar
- same stick may be used. fastest method, least prep
how are aerosols created when streaking
-from hitting the edge of plate with stick- send bacteria flying into air. (may also cause colonies to grow where there aren’t streak lines)
How to culture urine
- MSU (some contamination)
- UTI has a lot can be differentiated form normal urine
- Low amount of bacteria: normal urine, High amount indicates UTI
- Must be fresh no longer than 2 hours at room temp
- urine refrigerated as soon as collected ok for 24 hrs
- preservative used (boric acid)
dipstick and urine culture
-slides coated with media dipped into well mixed urine -slide is placed in container and incubated q
Calibrated loops and Urine culture
-small loop used 0.001mL
-loop immersed in urine -remove at 90 degree angle
-plate streaked
BAP and mac
carbon dioxide incubation
candle jar-classic -large jar used, candle put in, lighted, lid closed uses up all O2 and CO2 is released, jar is then incubated -gives 3%v/v atmosphere and moisture for bacteria
CO2 incubators also used
how are CO2 and O2 concentrations determined in carbon dioxide incubators
- FYRITE GAS ANALYSER
- sample of incubator air collected in a rubber bulb and aspirated into the FYRITE. concentration of carbon dioxide read from the scale on the instrument
anaerobic bacteria
can’t grow in presence of O2 - grow in body parts where little- to no oxygen. -o2 free culture media and transport needed
anaerobic jars
O2 removed and replaced by another gas (most removed by vacuums) or a catalyst - H2 and O2 combine to form water, the catalyst would be H2
catalyst will stop working when: enough moisture in air, moisture from hands if touching catalyst, hydrogen sulphide gas (produced from anaerobic bacteria growth)
how to properly use anaerobic jars
- jars not set up until enough plates
- plates go in jar with anaerobic indicator, catalyst and hydrogen supply
- jars go in incubator
QC with anaerobic jars
- water: if catalyst active and hydrogen present water will form
- Anaerobic indicator changes to colourless if anaerobic conditions obtained. methylene blue is blue but changes colourless when anaerobic. resazurin used and changes from pink to colourless when jar is anaerobic
anaerobic cabinets
best conditions for growing anaerobes
- completely sealed, planting and sub culturing is done in here. incubator in cabinet as well.
- all specimens and equipment enter cabinet through holding room- once oxygen removed it is moved into anaerobic working area
- gloves attached to front portal
Microaerophilic incubation
-small amount of oxygen required
-candle jar okay for some- best to use microaurophilic gas pack or compressed gas
-
campylobacteria
- truly microaerophilic
- can’t grow in ambient air or anaerobic atmosphere
- must be incubated in microaerophilic conditions
- some need temp 42-43 degrees and are incubated in separate incubator on Campy-BAP media
two most common methods to achieve microaerophilic conditions
- campy gas pak (they are put in jar with specimens)
- evacuation replacement- o2, co2 and hydrogen are used to flush out anaerobic jar plates put in plastic bag and air squeezed out and replaced by gas mixture and the bag is sealed and placed in incubator
what is gram stain used for?
primary purpose is to differentiate bacteria into two groups so physician can determine antibiotic type.
gram pos
stain purple blue or black
gram neg
stain pink or red
fixation of slides (4 things it does to cells)
- dry completely then fixed with methanol and it:
- causes bacteria to stick to slide
- kills vegetative bacteria (not spores)
- makes cells permeable to stain
- prevents autolyses or changes in cells
types of fixation
- heat fixation (low temp heating tray- not hot enough to kill or bunsen burners- passed thru flame)
- alcohol fixation- primary method- methanol** better than heat: cells preserved closer to true morphology
how are slides stained (after fixation)
- crystal violet
- iodine
this is when cells are stained purple-black - decolorization (most critical stage) - gram pos remain purpley blue and gram neg cells are decolonized
- counterstain safranin to stain gram negative cells to pink
why is decolorization important?
left on too long: over decolorization could occur and the stain would be removed from gram pos cells. if its not left on long enough under decolorization could occur resulting in a stain that is not removed from gram neg cells
what happens if slide over decolourized
-gram pos cells appear gram neg
background debris and other cells appear ok
gram neg bacteria or cells are not altered
under decolourization
gram neg appear to be gram os
background debris and other cells appear gram pos
gram pos bacteria not altered
how long are specimens saved for?
-refrigerated until culture completed and reported ( may be needed for re-culutre if growth not consistent with expected results)
virology
study of viruses
use electron microscope
how are viruses grown
tissue culture flasks using aseptic technique- antibiotic used to prevent bacterial growth.
*cells virus has infected will show diagnostic changes CYTOPATHIC EFFECTS
tissue cultures for virology
- store at -70
tubes coated with monolayer which are culture medium
how are viruses more conventionally tested for
serology- testing patients serum for presence of antibodies
molecular techniques- isolate viral RNA and DNA from patient samples, then amplify amount and perform further tests to identify virus based on nucleic acid sequences
mycoses
fungal infections
-can be grown on culture media and can form colonies
(less common than viruses and bacteria)
how long does it take for yeast to form colonies
24 hours
how long does it take for mold to form colonies
two to four weeks and incubated at room temp
what culture medium is used for fungi
sabouraud dextrose agar SDA
acid pH
low concentration of peptone high concentration of carbs
encourages fungal growth not bacterial growth
dermatophytes
multicellular fungi that attack keratin pcontaining structures- skin, hair, nails - cause superficial infections RINGWORMMM
specimens are skin scrapings
2-4 weeks for colony- incubated at room temp
Candida albicans 7
- colony forming yeast 24 hrs
- gram pos
- oval shape
- 3-5 times larger than bacteria
- form buds
- *part of normal flora can cause infection called CANDIDIASIS -from antibiotics, immunosuppressed patients, chronic diseases like diabetes
- treated with anti fungal
parasites in micro are from where
intestine
tow types of parasites
protozoa (unicellular)
metazoa ( intestinal worms)
trophozoite
protozoa parasite
- live and multiply in intestine
- inflammation and shallow lesions in gut wall, diarrhea, cramps, fever
- produce cysts in feces- these persist contaminate food and water
trichomonad vaginosis
protozoa parasite
- epithelial walls of vagina and urethra from men and women
- sex, toilet seats, bathtubs, back splash from a toilet
- vaginal itching, dysuria, discharge
- men usually asymptomatic
- flagellated protozoan exists only in trophozoite form
meazoa
intestinal worms such as pinworm or tapeworms. produce ova and are shed in stool- ova are morphologically distinct and are identified microscopically
pin worm
-common
-white 1cm in length
-ingestion of ova- eggs hatch in small intestine and migrate to large. woman migrate to anal region= itching which contaminates hands- reinfection cycle continues
-collection done by touching perianal skin with sticky pad to collect ova which are microscopically diagnosed
1 worm= 10,000 ova
Ascaris lumbricoides
large intestinal worm that resembles an earth worm.
- prevelant in tropics
- adult lives in small intestine -female =200,000 eggs per day -feces
macroscopic worms
pin worms, ascaris, whip worms and sections of tape worms
microscopic parasites
- ova
- protozoa trophozoites
- cysts
ova and parasite collection procedure
system 1
vial 1 5-10% formalin (preserves cysts and ova)
vial 2 Polyvinyl alcohol (PVA) preserves cysts and protozoa
system 2
one vial sodium acetate formalin preserves all
ova and parasite examination
- look for worms- applicator sticks sift thru stool
- pin and whip worms look like coarse white threads tapeworm segments look like undigested popcorn kernels - round worms submitted by patient usually - stained slides for protozoa and cysts then scanned microscopically
- concentration for ova and cysts using remainder of washed sediment
