lab techniques Flashcards
when should liquids be inoculated first and when after the solid?
first with a liquid medium
if a swab or stool sample, you inoculate liquid after the solid to avoid diluting the sample.
what to do if you have a great deal of liquid sample (more than 10mls)
centrifuge tto obtain the denser medium with more microbes - concentrate the medium. then you inoculate the concentrate
contamination with slides
avoid transferring organisms from a liquid medium to another liquid or solid medium or slide. The slide harbours bacteria since it is glass, so never place loop back into specimen after doing a smear. Therefore smears are done after sulture media
aseptic techniques (8)
- caps and lids should be kept in hand while sample is being processed
- caps and lids replaced as soon as possible
- plate lids placed facing upwards on benches and should be replced as soon as possible after inoculation
- production of aerosols: open slowly in biological cabinet
- never get close to plates, keep away from face and wear PPE
- disposable jar of lysol for loops
-forceps and scissors autoclaved and sterilized before and after use - disposable forceps and scissors used if possible and put in sharps bin
subculture from solid to liquid
select a representative colony or colonies of organism to be subcultured. use aseptic techniques to transfer to appropriate broth using a sterile disposable loop.
emulsify the organism using the inside of the container and agitate before incubation to distributeorganism through broth
subculture from solid to solid
choose representative colony with sterile disposabel loop, subculture on appropriate medium and streak
use for streaking
when you want to create seperate colonies to identify bacteria from a lot
streaking with urine sample: size of loop used, and relevance of growth
1 microlitre loop used. urine is a dilution of our sample so not all growth is relevant. for a specific bacterium to be relevant there have to be more than 100 colonies. If there are more than 2 types of bacteria with around a 100 colonies, the sample is retaken as it is said to be contaminated.
important notes when inoculating from liquid broth
never fully insert loop into media or else it will travel down and contaminate the rest of the loop including your hand, imp that it is swirled beforehand due to different atmospheric conditions at which they grow
use of swabbing
used to have confluent growth–> equal growth over the surface, can be used for succebtilibility testing via kirby bauer technique.
kirby bauer technique. how and why
swabbing for confluent growth, fix antimicorbial agent discs on the plate and incubate. presence or absence of growth around discs is an indirect measure of the ability of the compound to inhibit the organism.
used to determine the sensitivity or resistance of pathogenic bacteria to various antimicrobial agents. helps in sleecting treatment options
making anaerobic conditions
anaerobic jar: petri dishes, sachet with sodium bicarbonate and sodium borohydride, palladium pellets catalyst and anaerobic indictaor (methylene blue or strict aerobe). methylene bulue turns colourless in no oxygen.
anaerboic incubator- rich in N2 instead of O2
haemolysis colours on plate
beta haemolysis- white around dolony- all RBC destroyed
alpha haemolysis- green due to reduction of haemoglobin
alpha prime haemolysis - very slow beta haemolysis, seen that after incubation it starts to turn white
identification of bacteria
- microscopy: morphology and staining
- cultural grwoth characteristics: morphology, changes brought to culture medium (haemolysis, pigmentation), apibility to gorwo on specific media
growth on special media (selective and differential)
- biochemical characteristics (fermentation of sugars)
- serological tests
- analysis of metabolic end products
- molecular biology test
- growth characteristics : rapidity, atmospheric conditions, optimal temp
