CLM: Intro to Micro

KOH preparation for

- fungus/yeast in skin, hair, nails, and sputum specimens - thrush (Candida albicans)

India Ink Preparation for ID of ____ - what does it cause? - what do we see?

Cryptococcus neoformans (cryptococcal antigen in CSF) - Causes fungal meningitis - See capsule (C. neoformans is encapsulated) (also used for yeast in CSF)

Acid-Fast stain and culture for _____

mycobacteria

In PCR, a _______ is needed to match perfectly to the wanted gene

primer

India Ink Preparations for ____

yeast in CSF (cryptococcus neoformans)

Culture for Mycobacterium

- Culture for M. tuberculosis - can NOT culture M. leprae (Have no media that allows for growth of M. leprae)

Saline Wet Prep for ____

- Parasite (Trichomonas vaginalis) from vaginal secretions *Parasite still alive when stained* (can see flagella & undulating membrane)

what happens in KOH preparation?

- Specimen is mixed with KOH - Specimens softens, is digested (keratin destroyed) - Fungal elements are visible

Examples of Universal Precautions

- Wearing gloves, gown, mask, protective clothing - Washing hands - Proper disposal of waste in properly marked containers - Clean up spills - Use required daily housekeeping procedures - Not recapping needles - Laundry

General Specimen Collection - Protocol (3)

- be aware of special treatment when handling specific types of specimens (don't put CSF in fridge; maintain anaerobic specimens) - culture takes long time for lab to perform - proper specimen collection & handling required

describe streaking for isolation

- have dense growth in 1 quadrant; less growth in another quadrant, even less in another quadrant - gives us isolated colonies (know if norma flora vs infectious agent) - done on purpose

Culture of Anaerobic Bacteria

- requires special sample collection tubes & special equipment - Set up anaerobic conditions in a chamber/candle jar/CO2 packet (ex. Brewer jar)

Walk through the process of specimen collection starting at throat swab

1) Collect specimen → throat swab in area of throat that is red/inflamed; maintain sterility by putting in tube 2) take to lab --> setup culture in biological cabinet 3) streak for isolation 4) incubate 5) read culture plates for bacterial growth 6) further testing (antimicrobial susceptibility)

Process of PCR

1) Denaturation - DNA denatured (heating) → unwinds → 2 single strands 2) Annealing - Primers bind only partner to wanted gene - If primer doesn't bind → washed away 3) Polymerization 4) DNA replication

process of acid fast stain

1) Heat & Carbol Fuchsin (1º stain) - both pink 2) Acid Alcohol (decolorizing) - AF pink; non-AF = clear 3) Methylene blue (counterstain) results: AF = pink; non-AF = blue

General Specimen Collection Process:

1) Identification of Appropriate Media 2) Culture Set-Up 3) Agar Plates for Isolation of Bacteria (streaking for isolation) 4) Incubation of Bacteria 5) Reading Culture Plates for Bacterial Growth 6) Identification by Further Testing - Specific Media for Certain Bacteria - Biochemical Tests for ID - Antimicrobic Susceptibility (disk diffusion or automated method)

Types of Testing for Virus Infection (3)

1. Cell culture for viral growth 2. Amplification of virus-specific DNA (PCR) 3. Antibody testing of pt serum

Process of Gram Stain

1. Fixation (on glass slide) 2. Crystal violet (1º stain) 3. Lugol's iodine (2º stain) 4. Decolorization 5. Safranin (counter stain) results = GP = purple; GN = red

how many cycles of PCR do we run?

35 - exponential amplification (get 68 billion copies from 1)

_____ fold rise in titer strongly suggests resolution

4

results of acid fast stain

Acid fast = pink (retains color) non-AF = looks blue (loses color)

After decolorization and staining with safranin (counter stain), _____ organisms show up red

GN (safranin allows us to visualize GN organisms)

After being stained with crystal violet, ____ organisms is purple

GP

Results of Gram Stain

GP = purple GN = red

Infection - initial response - later response

Initial response is IgM - Increase followed by decrease Later response is IgG - Increase and plateau

Amplification of virus-specific DNA includes ____ methods

PCR

______ test for trichomonas

Saline Wet Prep for Trichomonas

body's response to virus; what occurs first (IgM or IgG)?

Short term = IgM - occurs first Long term = IgG - second

principle of universal precautions

Treat all blood/body substances as infectious

2 types of parasite testing

Wet direct preparation from fresh stool - For trophozoites Stool for parasite set-up and concentration - For cysts

in a _____ stain, mycobacteria retains the color, even under acidic conditions

acid-fast stain

Acute vs Convalescent Serum

acute = 1st (~1 week after infection) - high/rising IgM, no IgG convalescent = 2nd sample (~2 weeks later) - high/decreasing IgM, high/rising IgG

Gram stain and culture used for ____

bacteria

List and define the five categories of organisms important for Medical Microbiology.

bacteria viruses fungi archaea protists

Microbiology encompasses what 4 things?

bacteriology (bacteria) mycology (yeast, fungi) parasitology (parasites) virology (viruses)

mycobacteria

cell walls have mycolic acid in them (don't tolerate gram stain process) - TB (M. tuberculosis) - leprosy (M. leprae)

Universal precautions involves avoiding ____ at all times and using ___ technique

contamination; sterile

Kirby Bauer Disk Diffusion

discs with different antibiotic; positioned on plate of a single organism - look for zones of inhibition - bigger zone = more susceptible to antibiotic on disc - no zone = resistant/not effective - tells use sensitive vs resistant to antibiotic

KOH preparations and culture for ____

fungi/yeast (including thrush - candida albicans)

_____ are bacteria like TB and leprosy

mycobacteria

Antibody testing of patient serum for disease ______, _____, and _____

presence, progression, and resolution

PCR advantage and disadvantage

pro = very fast & specific; gives us exponential amplification con = not all viruses are set up for PCR diagnosis

IgG memory cells recognize and rapidly expand response

reinfection

_____ are insect vector-borne infections (fall under bacteria)

rickettsia

Need ____ for parasite testing

stool

motile parasites known as ______; convert to ____ when stressed (have thick cell membranes)

trophozoites; cysts

Any measure you take to protect yourself and others from acquiring/transmitting infectious agents

universal precautions

Cell Cultures, Ab Titers, Molecular Techniques (PCR) for _____

viruses

______, if present, infect cells and cause morphological changes to the cells (lysis)

viruses

viremia

viruses in the blood

The diameter of the ______ correlates to the sensitivity of a bacterium to an antibiotic

zone of inhibition