MICR5832 L3: Microscopy & Culture 84/25

What can we learn from clinical material?

-Inflammatory response/tissue damage

-Microbial complexity, quantity and quality

-Procedures for identification/cultures

-Presumptive or definitive identification

What can we learn from culture material?

-Staining reactions, microscopic morphology

-Primary guide for identification of pathogens/commensals

When are wet mounts performed?

-Microscopic morphology is distinctive

-Strong presumptive or definitive diagnosis

-Shape, size, motility

-Fungi and parasites good candidates

What are some clinical specimens that wet mounts are routinely used for?

1) Genital swabs

2) Candidiasis

3) Trichomoniasis

4) Fecal material

5) CSF

6) Urine

What are these wet mount specimens?

1) Ova, Cysts and Parasites (OCP) stained with Iodine

2) Stained with India Ink, associated with Cryptococcal Meningitis

3) Motile Trophozoites

4) Doesn't require stain to judge inflammation/quality of sample

5) Budding yeasts or Pseudohyphae

1) Fecal material

2) CSF

3) Trichomoniasis

4) Urine

5) Candidiasis

What microscope would you use to make a stained wet mount of fecal concentrates for OCP examination?

Bright field microscope

What microscope would you use to make an unstained wet mount?

Phase contrast microscope

What is this?

-Increases contrast of transparent objects

-3D view, topography

-Easier to see casts, cells, morphology

Phase contrast microscope

What does phase contrast microscopy help you to see?

-Cells

-Casts

-Morphology

What is this?

-Density increases with darkness

-Increases contrast but different image quality

DIC (Differential Interference Contrast) Microscope

What is this?

-Beyond resolving power of other light microscopes

-Used for thin-celled spirochaetes

-Background doesn't shine

Dark-field Microscope

What would you use dark field microscopy to view?

Thin-celled spirochete such as Borrelia burgdorferi

What type of specimen preparation is used for the majority of clinical specimens?

Stained dried smears

Why use stained dried smears instead of wet mounts?

-Morphology is not enough

-Lack of info about biochemical/physical properties

-Staining and biochemical tests required

What is this?

-Wright-Giemsa

-Iodine

-Trichrome

General stains

What is this general stain?

-Used for wide variety of cell types

Wright-Giemsa

What is this general stain?

-Faecal wet preps for intestinal parasites such as Giardia cysts

Iodine

What is this general stain?

-Fecal smears for intestinal parasites such as Giardia intestinalis

-Cytoplasm: Green

-Chromatin: Red/Purple

Trichrome

What is this?

-Gram

-Ziehl-Neelsen

-Kinyoun

Dye-Based selective stains

What selective dye-based stain is this?

-Cell wall structure used to distinguish 2 main groups of bacteria

Gram stain

What selective dye-based stain is this?

-Distinguish acid-fast bacteria such as Mycobacteria, Nocardia

-Stain binds to mycolic acid in cell wall

-Acid fast turns red, non acid turns blue

Ziehl-Neelsen

Kinyoun's stain

What are some acid-fast bacteria that could be identified by Ziehl-Neelsen and Kinyoun?

-Mycobacterium

-Nocardia

What is this?

-Uses UV illumination

-Auramine-Rhodamine stain

-Calcofluor White and Blankophor stains

Fluorescent selective stains

What fluorescent selective stain is this?

-Distinguishes acid fast bacteria like Mycobacterium tuberculosis

-Binding of fluorochrome to mycolic acid in cell wall

-Yellow, orange fluorescence

-More sensitive/cheaper than other stain, less specific

Auramine-Rhodamine stain

What fluorescent selective stain is this?

-Stain fungi and a few parasites such as Aspergillus

-Based on fluorochrome binding to chitin or cellulose in cell wall

-White fluorescence

Calcofluor White/Blankophor stains

What is this?

-Hemotoxylin and Eosin

Biopsy-selective stains

What is this?

-Biopsies all have this stain performed on them first

-Provides info about cell types to pathologist

-Basophilic: Blue

-Acidophilic: Pink

Hemotoxylin and Eosin stain

What is this?

-Romanowsky stains

-H&E stain

-Papanicolaou stain

Aspirate stains

What are some examples of Romanowsky stains that are used most commonly for aspirates?

-Diff Quick

-Giemsa

-Wrights

What aspirate stain is used specifically for cervical cytology, such as a Pap smear?

Papanicolaou stain

What are the steps for gaining info from microscopy?

1) Evidence of inflammation

2) Complexity of microbial flora

3) Quality of sample taken

4) Presumptive diagnosis (pre-culture)

5) Special procedures required

What leukocytes is a sample most likely to contain when searching for evidence of inflammation?

PMNs (Polymorphonuclear Leukocytes)

True or False: PMNs do not show up in a Gram stain

False, they are easily identified such as in a Gram stain of sputum

What are some less common leukocytes to see while searching for evidence of inflammation?

-Monocytes

-Macrophages

-Lymphocytes

-Eosinophiles

-Not normally differentiated in microbio lab

What does it mean if you find lymphocytes while searching for evidence of inflammation?

-Indicative of viral infection

-Found in CSF during meningitis (Wright's stain)

-Bacterial infections, chronic immune response

How do you determine complexity of microbial flora?

-Monomicrobial or polymicrobial infection

-Contamination with normal flora

What sample quality would you want for diagnosis of cystitis/nephritis?

Midstream urine

Why would midstream urine (MSU) be preferable for diagnosis of cystitis/nephritis?

Reduces contamination of urine with urethral flora

How can you tell a urine sample was improperly collected?

-High squamous epithelial cell count

-Contamination with normal flora from urethra, external genitals

What sample quality would you want for diagnosis of LRT infections?

Early morning sputum

How would you properly collect early morning sputum?

-Rinse mouth with water

-Deep cough directly into sterile container

How can you tell a sputum sample was improperly collected?

-High squamous epithelial cell count

-Heavy contamination with normal mouth flora

-Less likely to isolate LRT pathogen

What are some examples of presumptive diagnosis?

-Budding yeasts & pseudohyphae in genital swab (Candidiasis, genital thrush)

-High neutrophilia in CSF (Bacterial meningitis)

What is this?

-Empirical therapy

-Therapy based on a clinical educated guess

-Incomplete information, no definitive diagnosis

-Typically broad-spectrum antibiotics

Why would you need special procedures to identify a specimen?

-Acid fast bacilli in sputum, possibly Mycobacteria tuberculosis

-Löwenstein-Jensen media required

-Weeks incubation period

-Biohazard cabinet to protect handler

-No washing in tap water or environmental Mycobacteria will produce false positive result

What special medium is required to incubate Mycobacteria tuberculosis for weeks?

Löwenstein-Jensen Medium

What are some examples of culture characteristics?

-Elevation (shape), margin (edge), density (opacity), size, color, consistency, diffusible pigments

-Odor and growth on selective media

-Appearance on differential media

-Optimal growth temperature/oxygen level

What bacteria have brittle consistency?

-Nocardia

-Cause wound infections

-Chalky, dry, crumbly, wrinkled

What bacteria have waxy consistency?

-Corynebacterium and other diphtheroids

-Actinomycetes

What bacteria have a butyrous/butter-like consistency?

-Mucoid bacteria that heavily produce EPS in their capsule

-Very common: Staphylococcus

-Klebsiella, Enterobacter and other enteric Gram Negs

-Strep pneumoniae

-Ps. aeruginosa (some)

What are these bacteria based on their odors?

1) Old sock

2) Fruity, grape-like (this is a LIE)

3) Putrid

4) Musty, mouse nest

5) Freshly-ploughed field

1) Staph aureus

2) Ps. aeruginosa

3) Proteus mirabilis

4) Hemophilus species

5) Nocardia

What temperature do most bacterial pathogens grow at, especially if they are skin pathogens like acne bacteria?

37C

What temperature does Campylobacter jejuni grow at?

42C

What bacterial species grow at 0C?

-Listeria monocytogenes

-Yersinia enterocolitica

What bacterial species are capnophilic and grow at 5% CO2?

-H. influenzae

-Strep. pneumoniae

-Neisseria meningitidis

What bacterial species are anaerobic and grow in the absence of O2?

-Bacteroides

-Clostridium

What bacterial species are microaerophilic and grow in low O2?

Campylobacter

What is a medium that is both differential and selective?

-Selects for Gram Negative bacteria

-Differentiates lactose fermenters vs nonfermenters

MacConkey Agar

What is a medium that is both differential and non-selective?

Blood Agar

What type of organisms are encouraged to grow in a non-selective agar with growth enhancers added to it (enriched media)?

Fastidious organisms

What is this?

-Used as a supplement to agar plate culture

-Used to detect small numbers of microbes

Broth medium

What is this?

-Liquid medium designed to encourage growth of organism present in small numbers

-Suppresses other organisms present

Enrichment broth

What medium would you use ideally to detect anaerobes?

Thioglycollate broth

What non-selective enriched is this?

-Rich nutrient medium from sheep, horse

-Supports growth of some (not all) fastidious organisms

Blood Agar

Why is Blood Agar considered a differential medium?

Hemolysis can tell us what type of pathogen it is

True or False: Hemophilus influenzae does not grow on Blood Agar

True

What non-selective enriched media is this?

-Blood agar in which erythrocytes lysed by heat

-Erythrocyte lysis turns medium brown

Chocolate Agar (CHOC)

What factors are released from erythrocytes in Chocolate agar to support Hemophilus growth?

-X Factor: Hemin

-V factor: NAD

True or False: Hemophilus influenzae can grow on Chocolate Agar

True

What selective media is this?

-Acids in blood agar select against Gram Neg bacteria

-Successful Growth: Implies organism is Gram Positive

-H. influenzae cannot grow on this agar (no X and V factors)

Colistin-Nalidixic Acid (CNA) Agar

What bacteria would you use CNA agar to select against (inhibit growth)?

Hint: Contains Colistin and Nalidixic Acid

Gram Negative

What selective media is this?

-Antibiotic in agar selects against Gram Positive

-Contains X and V factors to support H. influenzae

-Successful Growth: Implies organism is Gram Negative

Bacitracin Chocolate Agar (BCA)

What bacteria would you use BCA agar to select against (inhibit growth)?

Hint: Contains Bacitracin

Gram Positive

True or False: Hemophilus influenzae can grow on Bacitracin Chocolate agar

True

What selective and differential media is this?

-Bile salts and crystal violet inhibit growth of most Gram Positive

-Lactose and pH indicator allow differentiation of Gram Negative

MacConkey Agar

What color is MacConkey Agar if there are lactose fermenters present to use lactose as a Carbon source?

Pink/red colonies from acidic products

What color is MacConkey Agar if there are no lactose fermenters present to use lactose as a Carbon source?

Colorless beige colonies

What bacteria would you use MacConkey agar to select against (inhibit growth)?

Hint: Contains Bile salts and Crystal violet

Gram Positive

What selective and differential media is this?

-Eosin Y and methylene blue inhibit Gram Positive

-Acidic products from fermentation of lactose or sucrose differentiate Gram Negative

Eosin-Methylene Blue (EMB) agar

What bacteria would you use EMB agar to select against (inhibit growth)?

Hint: Contains Eosin Y and Methylene Blue

Gram Positive

What color is EMB Agar if there are lactose fermenters present to use lactose as a Carbon source?

Black/dark purple colonies with green metallic sheen

What color is EMB Agar if there are no lactose fermenters present to use lactose as a Carbon source (EX: Sucrose)?

Translucent colorless or light purple colonies

Name an example of a clinical sample that would be inoculated onto both nonselective and selective media because the site of infection passes through/is found at a site with normal microbial flora

-Respiratory tract (throat/mouth/sputum)

-Genital tract

-Intestinal tract (faeces)

-Skin (& wounds)

Name examples of samples from normally sterile sites that do not require inoculation onto selective media unless somehow contaminated by normal flora

-Cerebrospinal

-Synovial

-Pleural

-Peritoneal fluids

What type of media would you use for these samples?

1) Fastidious organisms from normally sterile site or site where fastidious organisms are expected

2) Enteric Gram-negative bacilli

3) Mixed Gram Positive and Gram-Negative bacteria found

4) From sterile body fluids, tissues, lesions, wounds, abscesses

5) Fecal samples

1) Enriched medium

2) Selective and differential

3) Selective media for Gram Positive organisms

4) Broth medium as supplement

5) Additional selective media or enrichment broths

Where is the biggest bacterial colony ever?

Hint: Morning glory pool

Yellowstone

The best stain to use for detecting mycobacteria is:

A. Haemotoxylin and Eosin

B. Papanicolau stain

C. Ziehl Neelsen stain

D. Giemsa stain

Ziehl-Neelsen stain

Gram Positive bacteria are inhibited in a MacConkey agar by:

A. Methylene blue

B. Colistin

C. Bacitracin

D. Crystal violet

Crystal violet

Describe the difference between selective and differential culture media and give an example of each

Selective media inhibits the growth of certain microorganisms while promoting the growth of others.

Differential media allows for the visual distinction of different microorganisms based on their biochemical properties.

Selective: Mannitol Salt Agar, selects for Staph

Nonselective: Blood Agar, not used for feces since you want to isolate the fecal pathogen