Lab Techniques Flashcards
Specimen Handling for Mycology
use a biological safety cabinet level 2
* All specimens must be transported & processed immediately – fungus take a long time to grow – may delay patient results
-dont open the lid and wrap in parafilm to send out
DIRECT MICROSCOPY
KOH preparation:
- drop of 10-20% KOH on a glass slide
- Place nail or skin scraping or thin tissue piece into the drop and add a coverslip
- Optional-(Gently heat and allow to cool for 15 min)
- KOH & heat break down keratin and skin revealing fungal elements
- Can also use Calcofluor white & examine with fluorescence
DIRECT MICROSCOPY
India ink *** know how it looks know what a positive reaction looks like
Tissue stains:
India ink
* examine CSF/body fluid for Cryptococcus capsules
* 1 Drop of India ink + drop of specimen with coverslip
* Examine under 40X - wet prep brown mix
* The capsule repels the ink and a halo appears around the yeast
-yeast is the small cell in the big cell
Tissue stains::
- Periodic acid Schiff, Hematoxylin &
Eosin, Gomori methenamine silver & Giemsa
* Done directly on tissue, help visualize yeast or other fungal structures in tissue
Culture whats in it
- peptones & dextrose
- Sabouraud dextrose agar (SAB)
- SAB +antibiotics
- Potato dextrose agar
- BHI with blood + antibiotics
- Chromogenic agar used to differentiate Candida species.
- Gentamicin or chloramphenicol & cycloheximide added for selective purposes it inhibits growth of bacteria while cycloheximide inhibits saprophytic fungus that are contaminants
- Selective media not required for sterile specimens
- Selective media used for skin & nails for dermatophytes
Incubation of media:
* RT is optimum for fungal growth
* If suspecting a possible dimorph - also incubate a plate at 35C (yeast)
* Plates examined 2X a week for 4-6 weeks
- Macroscopically examine surface and reverse of colony and describe:
Front: colour and texture
* Specific colors seen
* Textures: wooly, cottony, velvety, powdery, waxy, suede, leathery
Reverse: colour
* Must record the number of days it took to grow, number of days till fruiting bodies appear, whether yeast or mold is recovered and which media and temp the fungus grew on
microscopic exam of growth from culture using a slide culture
Tease mount with Lactophenol Cotton Blue (LPCB)
-drop of media on glass slide
-using a bent needle pick up a colony and put it in the drop of LPCB
-tease the colony and coverslip. Can also use cellophane tape. Allows you to see the natural, undisturbed arrangement of micro and macro conidia
yeast wet prep and stain
wet prep
org + saline - look for budding yeast
gram stain
-stains gram positive - dont say its GP because it doesnt apply due to its cell wall with chitin. The yeast look HUGE under mic
-round to oval budding yeast cells with or without pseudo hyphae - very elongated cells
Yeast on culture *****
-most yeast are facultative but better aerobically so youll see it on BA and CNA
-grown for 18-24 hours but you wont see it on your ANO2 or MAC (pinpoint)
-yeast are white, small buttery
-cryptococcus are grey and mucoid due to its capsule
-candida looks like small, white can show feet on blood (looks like tentacles). if you see feet then you have to call it FAC Ana
-staph NH epi vs yeast is that yeast will not show up on ana
STAPH IS SMALL YELLOW
germ tube***** know what it looks like
-this is the beginning of the formation of a TRUE hyphae - filamentous extension from a yeast cell
-germ tube cells dont have construction at the base so they all attach to the mother cell
-If a constriction is present at the base of a germ tube, then it is pseudogerm tubes – beginning of pseudohyphae
* Provides a presumptive identification.
* Non sterile site: GT + = presumptive as C. albicans.
* Sterile site: GT +, identification must be confirmed because C. dubliniensis is also GT +
* GT negative – not C. albicans
-inoculate bovine serum with colony and incubate 2-3 hours at 35-37
germ tube positive - a tube that comes out of the cells ———O
germ tube negative oooooooO
chlamydospore production*** know the picture
with oxgall agar
-pick a colony and scratch the colony twice with straight wire and then streak
-put a coverslip on it since reduced oxygen exposure enhances production of chlamydospores
-creating a stressful environment to create chlamydospore
-look like tree branches or lollipops only by C.ablicans
-incubate at room temp over night and look with a microscope
urea***
-ability to produce urease
-urease hydrolyzes urea to ammonia and carbon dioxide
If urea in the broth is degraded ammonia is produced
▪ An alkaline environment is created, and the media
turns pink (phenol red indicator)
CRYPTO is urea positive
parasitology lab prep microscopic tech
wet mount
within 30 mins of collection
10% formalin
clear nail polish can be used to seal the coverslip or add in iodine to the saline but it wont show motility
- fresh stool, stool in 10% formalin or SAF – PVA gets cloudy with saline so can’t be used.
-very thin prep contained in the coverslip
-Can add iodine to stain parasites but kills trophs
worm helmiths eggs can be seen under 10x with battlement pattern
amoeba cysts under 40x
parasitology lab prep microscopic tech
concentration procedure
-used to concetrate parasites in feces and remove as much debris as you can
-done on fresh stool or with 10% formalin in SAF
1-Sedimentation method- formalin ether acetate
-used most often
-solution of lower specific gravity than parasites as they sink to the bottom of the tube as they are heavy and the debris floats on the top
-filter through sieve and is centrifuged to make layers ether- detritus-saline-sediment with parasite
-destroys trophozoite stages
-harder to see parasites as so much debris is removed
-thick and thin slide albumin is used to fix to slide and itll create thick and thin smears .
Ether or Ethyl acetate is used as an extractor of debris and fat
2.Sugar flotation - zinc sulfate or Sheathers sugar
-these solution which have high specific gravity than the parasites so they rise to the top and debris to the bottom
-some parasites dont float, some operculated eggs may open and cysts can get distorted
parasitology lab prep microscopic tech
permanently stained slides
critical examination of cysts and trophozoites
- shows nuclear detail, size and internal structure
Iron Hematoxylin: used for all parasites
Trichrome stain: used for all parasites
Kinyoun (Modified Acid fast)- acid fast bacteria
Calcofluor White: fluorescent dye used only as a SCREEN for parasites
Iron hematoxylin
Traditional stain for protozoan parasites.
* Oxidized hematoxylin (hematein) reacts with ferric
ammonium sulfate to produce iron-hematoxylin complex which is a basic dye.
* The iron hematoxylin stains the nuclei and chromatin of protozoan parasites
* used to stain regressively, i.e the slides are first
overstained with hematoxylin.
* Then decolorized using picric acid which removes the hematoxylin from background
Organisms will appear bluish or grayish with black
nuclear structures.
* Background will appear a pale blue-gray
trichrome
Uses 3 acid dyes that will provide contrast of various microscopic structures depending on which structures they stain
* Cytoplasm of trophozoites blue green
* Cysts purple.
* Nuclei and inclusions (chromatoid bodies, red blood cells, bacteria) and Charcot-Leyden crystals have a red color
* The background material usually stains green
scotch tape prep
- Specimen should be taken first thing in the morning
before the patient has bathed or had a bowel
movement - Commercial pinworm paddles OR
- Scotch tape prep using tongue depressor
- Press to perianal folds
- Press sticky side of tape to slide and examine
- Scan under low power for eggs and possibly adults
- Use caution Ova are infectious
vaginal or urethral discharge
- Fluids or swabs
- For live, motile Trichomonas
- Examine immediately
- Place fluid (or dab swab) on slide and coverslip
- Examine microscopically x40
- Look for darting motility
- Commercial culture media available to enhance survival
automated methods
immunologic diagnosis
molecular diagnosis
- Parasites that invade tissue are the ones that stimulate Ab production -can be diagnosed by serology
- EIA use monoclonal Abs to look for parasite Ag in the specimen
- Direct fluorescent uses monoclonal Ab’s to look for Ag in specimen
PCR OR MULTIPLEX OR MALDI
how to use level 2 BSC
- divided into two types (A and B) on the basis ratio of air recirculated and air that is exhausted
-turn off UV, start blower, purge for 10 mins, clean with 70% alcohol
-sash at 18 inches, minimize arm movement to avoid disrupting airflow, work clean to dirty, biohard bag should be inside
-no bunsen burner, aerosol generating instruments in the back of the bsc
-turn on UV after cleaning when done
cleaning a spill in the bsc
-keep running for 5 mins to clear the aerosols
-paper towel cover, put disinfectant or 10% bleach if blood , stand for 20 mins, throw away, clean with 70% alcohol and purge for 5-10 mins
sterilizer indicators
Biological
-dry spore paper strips
-glass ampoules in nutrient broth
-attest system with biological indicators
chemical - autoclave
-dyes in labels or tape with color change
physical - temperature recording chart
sealed glass ampoules
spores and media (tryptic soy broth)
-two tubes - one is processed in the load and the other is not
-break and incubate
-examined for growth
- Ampule that went through sterilization should remain the same colour – no change
- Growth control should change colour
good - you have realistic assessment of sterilization
bad- takes too long
Attest system - biological indicators
autoclave spore strip- media is purple incubate for 2 days @ 55 if bacteria grows then yellow -Geobacillus
ETO sterilizer spore strip - starts green, incubate for 2 days @ 35 if it turns yellow then bacteria grew
Paper with spores can be used. Put in Tsoy and incubate. turbidity means growth and clear means org is dead
chemical indicators
autoclave tape
changes color after autoclave - no guarantee of sterility
