LABORATORY Flashcards
GRAM STAINING
stain with (?) then gently wash with water.
Use (?) then gently wash with water.
Decolorize with (?).
Counter stain with (?) then gently wash with water.
Crystal Violet for 2 minutes
Lugol’s lodine for 2 minutes
Acid Acetone for 10 to 15 seconds
safranin for 45 seconds to 1 minute
MANNER OF REPORTING:
Smear shows «(?)»«(?)»«(?)»«(?)»«(?)»«(?)»«(?)»
(If present: smear is positive for intracellular/extracellular gram negative diplococci.)
occasional/few/moderate/many
gram’s reaction and/or yeast cells
bacilli/ coccis in singles/pairs/ chains/ clusters
occasional/few/moderate/many
occasional/few/ moderate/many
epithelial cells were also seen
ACID FAST STAINING
Stain with (?) then gently wash with water.
Use (?) for and allow it to steam and then gently wash with water (Do not let the stain boil).
Decolorize with (?).
Counter stain with (?) then gently wash with water.
carbol Fuchsin for 5 minutes
heat
Acid Alcohol for 10 to 15 seconds
Methylene Blue for 30 seconds
ACID FAST MANNER OF REPORTING:
(- or AFB NOT SEEN):
(+/- or DOUBTFUL):
1+:
2+:
3+:
4+:
0 AFB seen per 300 fields
1-2 per 300 fields AFB seen
1-9 per 100 fields AFB seen
1-9 per 10 fields AFB seen
1-9 per Field AFB seen
> 9 per field AFB seen
KOH MOUNT
1. Place [?] on a clean glass slide.
• For soft samples, let it stand for [?].
• In case of nail clippings, dissolve it first in [?] in a small test tube and stand for [?] before placing it on a slide.
2. Place a small amount of specimen and mix.
• Pass the slide [?] over a low flame in a bunsen burner. Do not overheat. Overheating will cause crystals to form.
3. Cover with coverslip.
• Apply gentle pressure using a [?] to disperse the specimen and place in a moist chamber to prevent specimen from drying.
4. Stand for [?].
• Allow the clearing of specimen.
5. Examine and report results.
• Examine under the microscope with reduced light source in [?] and shift to [?].
1-2 drops of 10% KOH
5 minutes
20% KOH ; 20-30 minutes
2-3 times
pencil eraser or forceps
5 minutes
LPO ;
● Maintaining an accurate inventory of all materials in the laboratory. This includes tracking incoming and outgoing materials.
MATERIAL MANAGER
● Ensuring that the supplies are properly stored.
MATERIAL MANAGER
● Identifying any potential shortages in supplies.
MATERIAL MANAGER
● Making sure that all the materials, equipment, and reagents used are back to their proper places.
MATERIAL MANAGER
● Collecting dirty glasswares and materials.
WASHER
● Washing glasswares and other materials used during the laboratory activities.
WASHER
● Sterilizing glasswares using an autoclave.
WASHER
● Storing clean glassware and other materials in a designated area.
WASHER
● Cleaning the laboratory spaces.
HOUSEKEEPER
● Making sure that wastes are properly segregated and disposed.
HOUSEKEEPER
● Turning on the UV lamp 15 minutes before entering and leaving the laboratory.
SANITATION
● Disinfecting the table tops and other laboratory spaces.
SANITATION
● Monitoring and logging the temperature of the room as well as the laboratory equipment in the Microbiology section.
MAINTENANCE
● Turning on the exhaust system upon entering the laboratory and turning it off before leaving.
MAINTENANCE
● Reading and following standard operating procedures (SOPs) for media preparation.
MEDIA PREPARATION
● Mixing contents according to SOPs.
MEDIA PREPARATION
● Sterilizing media using the autoclave.
MEDIA PREPARATION
● Storing media in a cool, dark place.
MEDIA PREPARATION
● Proper dispensing of media.
MEDIA PREPARATION
● Troubleshooting media problems.
MEDIA PREPARATION
MICROBIOLOGY LABORATORY WORKFLOW • SPECIMEN COLLECTION AND TRANSPORT
• PRE-ANALYTICAL TESTING
• GRAM STAINING
• CULTURE & BIOCHEMICAL TESTING
• IDENTIFICATION
• MICROSCOPY
• ANTI-SUSCEPTIBILITY TESTING
• REPORTING
DAY 1
Media Preparation of culture plates
Cooking of biochemical agar
DAY 1
Media Preparation of culture plates
Cooking of biochemical agar
DAY 2
Streaking of unknown
Primary gram staining of unknown
Incubation of primary plates and sterility plates
DAY 3
Colony morphology interpretation
Re-isolation of 2 colonies
Secondary gram staining
Reading of sterility test day 1
Washing of primary plates done reading
Save MAC for AST (hood)
DAY 4
Reading of Biochemical tests and Identification of organism
Anti-susceptibility testing with the available antibiotics
Repeat biochemical test
Reading of sterility test day 2
Washing of done biochemical tests
DAY 5
Reading of AST and repeated biochemical tests
Finalization of quota sheet on organism identification
DAY 6
Microscopic procedures using available slides (AFB, Vaginal and Penile discharge)
KOH preparation
LAST DAY
Practical exam and submission of quota sheets
Culture media According to Function:
Culture media According to Consistency:
: Supports growth of most non-fastidious bacteria •
• Supportive
● Ex: Nutrient agar, trypticase soy agar •
Supportive
: Contains added growth factors, e.g.. blood, vitamins, yeast extract
Enriched
Ex: SBA, CAP, brain-heart infusion, buffered charcoal-yeast extract agar
Enriched
: Contains additives such as dyes, bile salts, alcohols, acids or antibiotics
Selective
inhibit growth of certain bacteria
Selective
● Ex: ENA, EMB, MacConkey, HE, xylose lysine deoxycholate (XLD); Thayer-Martin
Selective
: Formulated to provide distinct colonial appearances based on certain biochemical reaction (lactose fermentation, HaSproduction)
Differential
● Ex: EMB, MacConkey, HE, XLD
Differential
: encourage growth of desired microbes by providing special growth conditions or added growth factors.
Enrichment Media
They are liquid in nature
Enrichment Media
● Ex. APW, Selenite Fluid, Thioglycollate
Enrichment Media
: observe colonies and pure growth: 2-3%
• Solid
: for motility: 0.5-1%
• Semi-solid
: no solidifying agents; growth within 4-6 hours
• Liquid
Grow in the presence of atmospheric free O2
Obligate aerobe
21% O2 + 0.03% CO2
Obligate aerobe
Cannot grow in the absence of air
Microaerophile
5-10% O2 + 8-10% CO2
Microaerophile
Campylobacter: Helicobacter
Microaerophile
Grows in the absence of O2
Anaerobe
5-10% H2 + 5-10% CO2 + 80-90% N2 + 0% 02
Anaerobe
Cannot grow in the presence of O2
Obligate anaerobe
Clostridium; Fusobacterium Actinomyces; Provotella
Obligate anaerobe
Capable of growth with or without O2
Facultative anaerobe
Enterobacteriacea, Staphylococcus
Facultative anaerobe
Does not grow well but survives in the presence of O2
Aerotolerant
Lactobacillus; Acrobacter
Aerotolerant
Grows in enhanced or enriched CO2 *cancle jar (3% carbon dioxide) or carbon dioxide incubator, jar, or bags
Capnophilic
5-10% CO + 15% O2
Capnophilic
Streptococcus: Haemophilus; Neisseria
Capnophilic
Lactose fermenter:
• Escherichia
• Klebsiella
• Enterobacter
Late Lactose Fermenter:
• Citrobacter
• Serratia
• Hafnia
• Yersinia
• Salmonella Arizona
• Shigella sonnei
H2S Producer:
• Proteus
• Citrobacter
• Salmonella
• Edwardsiella
Non-Lactose Fermenter:
• Serratia
• Proteus
• Morganella
• Providencia
• Yersinia
• Shigella
• Edwardsiella
• Salmonella
• Citrobacter (some)
PAD (+) and Urease (+):
• Proteus
• Providencia
• Morganella
