Clinical Microbiology Lab Exam #1 Study Notes

What are some qualities in our media/cultures that would propagate bacterial growth, isolation, and identification?

1. Nutrition: proteins, vitamins, minerals, carbohydrates, peptone and meat extract

2. Energy: carbohydrates (mostly glucose)

3. Essential minerals: present in peptone and meat extract

4. Buffering agents: optimum pH, amino acids, phosphates, citrates, bicarbonate

5. pH indicators: variation in pH of the medium by color change; phenol red

6. Selective agents: growth of only specific bacteria; antibiotics, tellurites, azides and bile salts

7. Solidifying agents: medium in solid or semi-solid state; agar or gelatin

8. Specific substances such as growth factors, enzymes may be incorporated into the medium for specific bacteria

What is the general purpose of supportive media?

Encourages the growth of most microorganisms; nutrient broth/agar, thioglycolate broth, trypticase soy broth

What is basal media?

a nutrient broth and peptone water utilized to culture bacteria; common in research laboratories

What is complex/enriched media?

a mix of many nutrients, utilization of peptone (degraded proteins from beef, milk powder, soy). Commonly utilized in the clinical laboratory (BAP, CAP, MAC)

What is defined media?

Defined media is known carbon, nitrogen and amino acids added as requirements to grow a specific type of bacteria. Utilized the growth of unusual organisms; vitamin, pH, nutrient requirement.

What are the primary plating media? Why are they considered primary?

Blood agar plat (BAP), Chocolate agar plate (CAP), MacConkey agar plate (MAC). These are all used in almost all routine cultures as they're complex media and result in growth of most common human pathogens.

What is the difference between selective and differential media?

Selective is utilized in growth of particular "selected" organisms while suppressing the growth of other organisms. Differential is used to provide the ability to visibly "distinguish" one organism type from another by using biochemical properties.

What is BAP?

enriched with trypticase soy base (soy peptones), 5% sheep red blood cells, NaCl.

It is a differential media by lysis of red blood cells.

1. Beta-hemolysis: complete lysis of RBC, clear zone around colony. (Strong: read print through plate, Weak: zone of clearing)

2. Alpha-hemolysis: hydrogen peroxide produced by bacteria oxidizing hemoglobin producing methemoglobin (greening of plate)

3. gamma-hemolysis: lack of hemolysis

What is CAP?

Chocolate Agar Plate: enriched agar only; warm sheep blood that have been lysed (releasing heme (X factor) and nicrotinamide adenine dinucleotide (NAD, factor V)); also has peptones and NaCl.

Grows almost all bacteria, especially fastidious organisms (Haemophilus sp. and Neisseria sp.)

What is MAC?

MacConkey Agar Plate: selective and differential media with peptone, dyes, bile salts, neutral red (pH indicator) and lactose.

Selective for Gram-negative bacilli: Crystal violet and bile salts prohibit growth of gram-positive organisms.

Differential for lactose utilization: lactose and neutral red allow for ability to differentiate organisms that can degrade lactose; pink when pH < 6.8; carbohydrate degradation bioproducts are acidic; LF (pink)/NLF(clear)

What is EMB?

Eosin-Methylene Blue Agar plate: Selective and differential and has peptone, eosin, ethylene blue, lactose, sucrose

Selective for Gram-negative bacilli: methylene blue prohibits growth of gram positive organisms.

Differential for lactose and sucrose utilization:

1. Eosin allows the ability to differentiate organisms that can degrade lactose. (LF - pink, NLF - clear)

2. Metallic green sheen is an indicator of lactose and sucrose fermentation ability of typical E. coli, Citrobacter, Klebsiella.

What is HE?

Selective and differential designed for isolation of Salmonella sp. and Shigella sp; Has peptone, yeast extract, lactose, sucrose, salicin, bromothymol blue, acid fuchsin, iron ammonium citrate, bile salts

Selective for gram negative bacilli: bile salts prohibit growth of gram-positive organisms

Differential for lactose and sucrose utilization:

1. Lactose, sucrose, and salicin allow for the ability to differentiate organisms that can degrade these products - Yellow/orange colony production.

2. H2S production from iron, differentiates Salmonella sp. --> black colonies

3. Non-lactose/sucrose fermentation - clear colonies

What is XLD?

selective and differential deisgned for isolation of Salmonella sp. and Shigella sp.; has yeast extract, xylose, lactose, sucrose, L-lysine, sodium chloride, sodium deoxycholate, sodium thiosulfate, ferric ammonium citrate, and phenol red

Selective for gram negative bacilli: Sodium deoxycholate (bile salts) prohibits growth of Gram-positive organisms.

Differential for xylose, lactose and sucrose utilization:

1. Xylose, lactose, and sucrose allow for the ability to differentiate organisms that can degrade sugars vs. non-degradable - yelloe colony production.

2. Lysine when decarboxylated by Salmonella turns the pH from acid to alkaline - red colonies

3. H2S production from sodium thiosulfate and ferric ammonium citrate differentiates Salmonella s

4. Xylose/lactose/sucrose fermentation (yellow), Lysine decarboxylated (clear colonies); H2S production (Black colonies)

What are some media that prohibits growth of gram-negative organisms?

Phenylethyl alcohol agar (PEA) and Colistin Nalidixic Acid agar (CNA)

What is PEA?

Phenylethyl alcohol agar (PEA): selective for gram-positives; phenyl ethyl alcohol inhibits DNA synthesis in gram-negative organisms; 5% sheep blood with TSA base.

What is CNA?

Colistin Nalidixic Acid: selective for gram-positives; antibiotics inhibit growth of gram-negative organisms, allowing gram-positives to grow; 5 sheep blood with TSA base.

What is MSA?

Mannitol Salt Agar: Selective and differential for Staphylococcus sp.; has peptone, NaCl, mannitol, phenol red pH indicator

Selective: 7.5% NaCl prohibits the growth of almost all organisms.

Differential: mannitol is fermented by Staphylococcus aureus producing acid by-products resulting in changes in phenol red indicator to yellow (pH 6.8); Other Staphylocccus sp. and Micrococcus sp. do not ferment mannitol.

What is Bile esculin?

Selective and differential for the

isolation of identify and isolate

Enterococcus species and group D

Streptococcus

Selective for Streptococci

1. Sodium deoxycholate (bile salts)

prohibits growth of Gram-positive

organisms

2. Only organisms that can tolerate high bile concentrations will grow.

3. Results in BLACK COLONIES

Differential for esculin utilization:

Esculinase enzyme, hydrolyzes esculin

What is aseptic technique?

A set of specific practices used in

microbiology labs to prevent the contamination of sterile

media, pure cultures, and the surrounding environment.

How do we accomplish pure cultures via aseptic techniques?

Accomplished by sterile disposable loops/needles OR using

flame to sterilize a metal loop/needle.

- Heat in the incinerator until it turns bright red

- Cool the hot loop on the edge of media to cool and THEN touch a colony of organism or go into broth culture.

• Keeping petri dishes closed until inoculating.

• Do not create aerosols of organisms

What is the Four quadrant bacterial streaking methodology and why is it important?

Patient samples often contain many different bacterial isolates.

We need a process to be able to:

- Visualize all organisms present.

- Quantitate each organism present.

- Isolate each organism to establish pure cultures for further testing.

How can we identify bacteria with colony characteristics?

- Size

- Edge

- Elevation

- Color

- Capsule production (glistening, wet)

- Appearance (dry, powdery)

- Optical property

- Texture

- Hemolysis

What is optical property?

The bacterial colony also varies in optical properties. Based on the opacity, the

bacterial colonies are of the following types -

1. Opaque - Non transparent (cannot see through it)

2. Translucent - Semi transparent (partially can be seen)

3. Transparent - Can clearly see through it

4. Iridescent - Shows different color in light reflection

How can you identify bacterial growth in brother culture?

1. Obligate aerobes (growth at the top of broth)

2. Obligate anaerobes (growth at the bottom)

3. Facultative aerobes (growth at the top with some growth going towards the bottom)

4. Aerotolerant aerobes (growth dispersed all around the brother)

5. Microaerophiles (growth below the top of the broth)

What some basics of microscopy?

1. Compound Microscope

o Series of lenses

o Light source

o Magnification from 40x to 1000x 2. Usage

o Essential in Microbiology, Hematology, Urinalysis & Body Fluids

3. Handling

o ALWAYS use both hands

o One at the base

o One holding the arm

4. Before each use:

o Examine for proper storage and possible

damage

o Clean the eyepiece and make sure stage is

free of oil.

5. AFTER each use, ensure proper

storage:

o Clean oil from stage & objectives

o Lowest objective/empty objective in

vertical position

o Stage in lowered

o Dust cover is placed over scope

What is the total magnification achieved based on?

The combination of eye and objective lens.

Eyepiece = 10x

o Objectives

o 4x = scanning objective

o 10x = low-power objective

o 40x = high-power or high-dry objective (Does not require oil, visualize fine details of sample, body fluids, urinalysis)

o 100x = oil immersion objective (Oil improves resolution of the magnified image, prevents scattering of light rays, directing

upward through the lens, most intense light required)

What is the focal length?

The distance from the centre of the lens to the principal focus

What is the principle of the gram stain protocol?

The principle of the Gram stain protocol is the differentiation of bacteria based on the chemical and physical differences in their cell wall composition, specifically the thickness of the peptidoglycan layer.

What makes bacterial cell wall important for gram-staining?

Cell wall type effects how we see

bacteria under the microscope!

• Peptidoglycan-macromolecule that

makes up the cell wall. (Think metal fence, that holds in the

bacterial components)

Peptidoglycan-Negative vs. Positive

What are used in the gram staining test>

1. Crystal violet

o Primary stain/dye, purple

o Retained by organisms with a high concentration of

peptidoglycan in the cell wall

2. Gram's Iodine

o Mordant, binds dye to cell wall

o Certain strains of bacteria will retain the stain even after

decolorizer

3. Decolorizer (Ethyl alcohol 95%)

o Washes away stain in those organisms unable to maintain due

to concentration of peptidoglycan or because of cell membrane

lipid content.

4. Safranin

o Counterstain

o Needed to visualize organisms where crystal violet is washed

away.

o Other counterstains can be used e.g. 0.05% carbofuchsin

What results from the gram-staining test that allow us to differentiate bacteria based on cell walls.

Gram-positive bacteria have lots of peptidoglycan so stain (crystal violet crystal violet) is

held up....looks purple.

• Gram-negative bacteria only have a little peptidoglycan so the stain isn't held up & its washed away. Only see the color of the second stain ( safranin red safranin red)....looks

dark pink