Micro Lab Techniques Flashcards
Differentiate between a Direct Specimen, Indirect Sample, and Sample from site (w/ normal flora).
- Collection method
- Example
Direct Specimen:
- Pathogen is located in an otherwise sterile site
- Collect Surgically or Needle Aspiration
- E.g. deep abscess
Indirect Sample:
- Pathogen is located in a sterile site but must pass through a non-sterile site to collect the flora
- Expectorated Sputum, Voided Urine
- E.g. pneumonia
Site Sample:
- Sample collected is a mixture of normal flora and infectious agent
- Troat swabs, Stool samples, etc.
- E.g. Skin abscess for Staph
What is another name for Acid-Fast Staining?
- Ziehl-Neelsen
What is a wet mount?
- what disease has historically be diagnosed this way?
- You just take some cells scraped off (vagina, cheek) etc. and look at them under the microscope
- This can be used to Dx Bacterial Vaginosis
What does a Dark Field test usually look for?
- how is it done?
- Looks for Gram (-) spirochetes like T Pallidum
- Can Dx Syphilis
- Shine like at correct angle and the Spirochete will glow
What is the biggest challenge when trying to perform a culture?
You need a pure isolate to do this
What are the following agar differential/selective for:
- Blood Agar
- Mannitol Salt Agar
- Eosin-methylene blue agar
Blood Agar - selective only on basis of Hemolysis
MSA - selective for staph, differential for staph aureus
EMB - Selective for gram (-), differential for lactose met.
When is MacConkey agar often used?
- Selective/Differential?
- Often used in G.I. associated Infections
Selective for Gram (-)
Differential for Lac+ and Lac-
A patient presents with bloody diarrhea and some bacteria from stool samples is cultured on MacConkey agar with Sorbitol.
- Explain why this is done?
- What are they looking for?
MacConkey is selective for gram (-) organisms, usually from the G.I. tract
- Sorbitol allows for differentiation between E. Coli O157, which is often responsible for G.I. infections involving bloody stool and other strains of E. Coli.
- Most G.I. strains will form Red colonies, EO157 will be WHITE on the agar
What type of Agar would you use to culture Neisseria Species?
- what if a patient had meningitis?
Thayer-Martin agar = Chocolate Agar + Antibiotics
- If pt. had meningitis, then you would use Chocolate Agar WITHOUT antibiotics, because CSF should be sterile and we want to grow any bacteria we can
Give the steps in obtaining a standard blood culture.
- Cleanse with 2% iodine before puncture
- Obtain AT LEAST 3 10mL samples in 24hrs (at different sites)
- Add to rich growth medium
- Look for Turbidity and Anaerobes daily for up to 7 days
In what Situations would you draw blood for culture?
- Sepsis
- Endocarditis
- Osteomyelitis
- Meningitis
- Pneumonia
How is a routine throat culture performed if you were looking for strep?
- agar used?
- Gram stain? why or why not?
- Swab posterior pharynx and tonsils
- ROUTINE then culture on BLOOD agar
- Confirm ß-hemolytic species are strep by looking at Taxo A sensitivity
**NO gram stain because Step Viridans will also be present if its a throat swab
Someone is suspected of having aspiration pneumonia and you want to do a sputum sample, what kind of culture might you want to do (step by step)?
- why?
- how does this deviate from standard protocol?
- Be sure sample is SPUTUM and NOT saliva
- Gram stain the organism
- Culture organism on ANEROBIC MEDIUM
- we do this because if its aspiration pneumonia then a gram (-) is the most likely organism to sneak up from the G.I. tract.
- Standard protocol would be to use blood agar.
What is the process of drawing spinal fluid to send it to culture?
- Lumbar Puncture
- SEND TO LAB IMMEDIATELY
- Samples are centrifuged and analyzed microscopically
- CULTURE on Blood agar and Chocolate Agar with NO ANTIBIOTICS
Do you need an anaerobic culture when doing a stool culture?
- why or why not?
No, anaerobes are part of the normal flora
How do should urine cultures be done?
- is urine sterile?
- Plate used?
- Streaking?
- Quantitatively or Qualitatively done?
- Urine is sterile until it gets to lower GU so urine samples will have bacteria
- Urine should be collected with 1st pee of the morning and should be taken in mid-stream
Plate/Streaking:
BAP (anything) and EMB (gram -) plates should be streaked with a 0.001 mL calibrated loop
- These tests need to be quantitative because there will always be some amount of bacteria in them, however there is a threshold that indicated infection
What is the threshold for the amount of colonies that determine a positive UTI test?
> 100,000 colonies per milliliter
This corresponds to 100 colonies with a 0.001 mL calibrated loop
What kind of technique is used to culture viruses?
Cell-Culture Technique
How is a cell culture performed?
- Specimen is put into a transport medium that is centrifuged
- cell debris fall to the bottom and virus stays in the supernatant - Supernatant is treated with antibiotics to prevent bacteria from growing
- Add this Treated Supernatant to Squamous cells
- Pour the cells in to a monolayer in a dish
***CELL LYSIS = Presence of Virus
What indicates a positive antibody detection test?
Four Fold Rise in Specific Convalescent vs. Acute Titer
How is non-specific Syphilis Testing Done?
- what is the test called?
- what are the components?
- what indicates a positive test?
- Rapid Plasma Reagin (RPR)
- Venereal Disease Research Lab (VDRL)
- Beef Heart cardiolipin is conjugated to carbon particles and this clumps in the presence of antibody to T. Pallidum
How is specific Syphilis testing done?
Serum from person with suspected infection is treated with Fluorecent anti-human IgG, the fluorescent IgG will bind to your antibodies that are bound to the bacteria and it will glow
Why would you use an antigen Detection assay over an antibody detection assay?
Antigens are present at higher levels than antibodies early in infection so if we can detect the disease earlier with these tests.
How is a virus neutralization assay performed?
See Diagram
- Antibody specific for a virus is added to a serum containing the virus
- Control is created of serum w/o antibody - Squamous cells are added and a cell culture is performed
- The sample with with antibody will not die
- the sample w/o antibody will die
RAPID ANTIGEN DETECTION TEST see diagram to see how it works
RAPID ANTIGEN DETECTION TEST see diagram to see how it works
How is HIV testing done?
- Use one of several priliminary tests
- Murex Single Use Diagnostic System (SUDS) - looks for Ab and presence of p24
- OraQuick (home test) for saliva antibodies
- ALWAYS follow up with a Western Blot positive test is indicated by:
Positive: p24, gp41, and or gp120/160
Intermediate: some bands
Negative: no bands
How is direct probing (Hybridization) performed and what is it used for?
- used to diagnose Bacterial Vaginosis and others
- Basically performed like microarray
1. Denature speciment and add it to a tray that has single stranded DNA hanging off of it
2. Pairing will occur if the DNA matches
3. We can detect how much binding took place by adding biotin (or something that has affinity for dsDNA and not ssDNA
How do Nucleic Acid Amplification Tests (NAT or NAAT) work?
- Amplify nucleic acid from virus using PCR
2. Analyze Sequence that is specific to that pathogen
When are is NAAT useful?
why?
- can it be used to determine pathogen load?
When organism is difficult to routinely grow and isolate.
**Its becoming the Gold Standard of Dx
- Its pretty rapid
- Because of amplification its also pretty sensitive
YES, it can be used to determine pathogen load
Would you use NAT for BV?
NO, because in BV there is no need to and the disease is caused by normal flora and amplifying it causes you to loose the diagnostic info which is how much of an overabundance is there
How is load determined using NAT?
essentially you make a calibration curve
- you measure the point of inflection on the PCR growth curves and see at what cycle the infection point is hit
- you can tell the load by comparing to controls that were prepared and graphed in the same way
How does an enterotube work?
Lots of agars are placed in a little tube and the bacteria is sent through each agar via a central line through all of the agars. BACTERIA ARE DIFFERENTIATED ON THE BASIS OF HOW THEY METABOLIZE
- Each agar group sums to a number, the summated numbers from each group are then become a combination that can be put into a database to see what organism you’re dealing with.
What kind of organisms can reduce nitrate to nitrite?
- what are the reactions?
Gram (-) rods
- Para-arsanilic acid or sulphanilamide + NO2 –> Diazonium Salt
- Diazonium Salt + Tetrahydrobenzoquinoline –> Pink azo die
What does an esterase test look for?
- what are the reactions?
- Detects an Esterase produced by Monocytes, Neutrophils, and Eosinophils
Indolecarboxylic acid ester + esterase –> Indoxyl + acid
Indoxyl + Diazonium salt –> Violet Azole Dye
What is MALDI-TOF?
Mass Spec that separates particles on the basis of size after they are ionized.
- Each bacteria has a unique protein spectrum that will give a unique MALDI-TOF sequence
What is the time to result for:
- RADT
- MALDI-TOF
- qPCR
RADT: 5-20 min
MALDI-TOF: 1 hr
qPCR: 1-2 hr
What is the time to result:
- Std PCR
- Probe Hybridization
- Metabolite Analysis
Std PCR: 3-5 hr
Probe Hybridization: 6-12 hr
Metabolite Analysis: 1-2 days
What is the time to result:
- Bacterial Growth
- Viral CPE
- Seroconcersion (ELISA)
Bacterial Growth: 1-2 days
Viral CPE: Several Days to Weeks
Seroconversion (ELISA): 2-4 weeks
