Review for Lab Practical #1

MLAB 352Dr. Mohamed A. El-Sakhawy

Lecture Outline• Exam format• Biosafety• Staining• Four-way plate streak• Plate counts• Media types• Temperature classification• Oxygen classification• Control with chemicals and antibiotics

Format of the Lab Practical

Biosafety

General Biosafety Rules• NO food or drink in lab.• NO cosmetics in lab.• NO open-toed shoes in lab.• Lab doors will remain closed at all times.• NO mouth pipetting.• Use care when handling potentially hazardous materials.• Wear appropriate personal protective equipment at all

times.• Dispose of all microbiological materials in designated

orange biohazard bags.• Dispose of all sharps and glass in designated sharps

containers.• Notify the instructor immediately when there is an accident

or spill.• Notify the instructor of pertinent medical conditions.

When You Begin Work for the Day…• Wash your hands.

• Disinfect the surface of your lab table.

• Put on your goggles.

• Put on your lab apron.

• Put on gloves.

When You Are Finished for the Day…

• Appropriately dispose of all waste.• Disinfect the surface of your lab table.• Remove gloves inside-out and dispose in

biohazard waste.• Remove apron, fold inside-out, and store in

your drawer.• Remove goggles and store in your drawer.• Wash your hands before leaving the lab.

Staining

Types of Bacterial Stains

• Simple stain – Stains cells but not background– Heat-fixed (killed) bacteria

• Negative stain – Stains background but not cells– Live bacteria

• Differential stain – Stains only certain types of cells based upon properties

• Basic stain – Positive charge– Attracted to negatively-charged cell wall

• Acidic stain – Negative charge– Repelled by negatively-charged cell wall

Reagents of a Differential Stain• Primary stain

– First dye used in the staining process– Will initially stain all cells and then be removed from a

subset• Mordant

– Improves the ability of the primary stain to bind cells• Decolorizer

– Removes the primary stain from a subset of cells• Counterstain

– Second dye that stains decolorized cells

A Smear is Prepared by Spreading Bacteria on a Glass Slide

Figure 4.15

Heat-Fixing a Bacterial Smear

http://iws2.collin.edu/dcain/CCCCD%20Micro/Heat_fix.htm

Gram Stain

http://iws2.collin.edu/dcain/CCCCD%20Micro/gram_stain.htm

(primary stain)

(mordant)

(decolorizer)

(counterstain)

http://student.ccbcmd.edu/courses/bio141/lecguide/unit1/prostruct/grmix.html

Examples of Gram-Positive and Gram-Negative Organisms

GRAM-POSITIVE• Staphylococcus xylosus• Bacillus megaterium

GRAM-NEGATIVE• Escherichia coli

Capsule Stain

• Alcaligenes faecalis• Enterobacter aerogenes• Klebsiella pneumoniae• Serratia marcescens

http://iws2.collin.edu/dcain/CCCCD%20Micro/capsule_stain.htm

(primary stain)

(decolorizer/counterstain)

NO HEAT FIXATION!

Acid-Fast Stain

http://iws2.collin.edu/dcain/CCCCD%20Micro/acid_fast.htm

(primary stain)

(decolorizer)

(counterstain)

Figure 4.18

• Mycobacterium smegmatis

Endospore Stain

http://iws2.collin.edu/dcain/CCCCD%20Micro/endospore.htm

(primary stain)

(decolorizer)

(counterstain)

• Bacillus megaterium

Four-Way Plate Streak

Four-Way Plate Streak

http://iws2.collin.edu/dcain/4way.htm

Plate Counts

Viable Plate Counts from Serial Dilutions

• TNTC – Too Numerous To Count – More than 300 colonies

• TFTC – Too Few To Count– Less than 30 colonies

Calculation of CFU/ml for Urine Specimens

• Count colonies– CFU/ml = # colonies/calibrated loop volume

• Threshold: 1 x 105 CFU/ml

Media Types

Nutrient Agar (NA)

• Special features:– None

• Used for:– Culture of non-fastidious

organisms

http://commons.wikimedia.org/wiki/File:Agar_Plate.jpg

Sabouraud Agar/Broth

• Selective media• Special features:

– Acidic pH (5.5)

• Used for:– Culture of fungi (yeasts

and molds)http://iws2.collin.edu/dcain/CCCCD%20Micro/sabouraudagar.htm

http://www.jlindquist.net/generalmicro/dfnewgfbpage.html

Brain Heart Infusion (BHI) Agar

• Special features:– None

• Used for:– BHI agar deeps used to

determine oxygen classification

http://iws2.collin.edu/dcain/CCCCD%20Micro/bhitubes.htm

Sodium Thioglycollate Broth

• Special features:– Sodium thioglycollate –

Reduces O2 to H2O

– Resazurin (dye) – Pink when oxidized (O2 present), colorless when reduced (no O2)

• Used for:– Culture of anaerobic

organisms

http://iws2.collin.edu/dcain/CCCCD%20Micro/thioglycollatebroth.htm

Mannitol Salt Agar (MSA)

• Selective• Differential• Special features:

– 7.5% NaCl– Mannitol (fermentation

substrate)– Phenol red (pH indicator)

• Used for:– Growth of halotolerant

species (esp. Staphylococcus spp.)

– Distinguishing between Staphylococcus spp.

http://iws2.collin.edu/dcain/CCCCD%20Micro/normalskinflora.htm

S. epidermis

S. aureus/S. xylosus

Blood Agar• Differential• Enriched• Special features:

– Sheep’s blood (nutrients, hemolysis substrate)

• Used for:– Culture of fastidious organisms– Distinguishing between

bacteria by hemolysis patterns (esp. Streptococcus)

http://iws2.collin.edu/dcain/CCCCD%20Micro/hemolysis.htm

Chocolate Agar

• Enriched• Special features:

– Boiled sheep’s blood (nutrients)

• Used for:– Growth of fastidious

organisms (esp. Neisseria spp.)

– Distinguish Neisseria spp. using oxidase test

http://iws2.collin.edu/dcain/CCCCD%20Micro/normalfloranasopharynx.htm

http://www.medschool.lsuhsc.edu/microbiology/DMIP/bsoxid.jpg

Mueller-Hinton Tellurite (MHT) Agar

• Differential• Special features:

– Tellurite (metal)

• Used for:– Distinguishing

Corynebacterium spp. (diphtheroids) based on reduction of tellurite to tellurium (black ppt.)

http://iws2.collin.edu/dcain/CCCCD%20Micro/normalskinflora.htm

Columbia C-NA (CCNA) Agar

• Selective• Differential• Special features:

– Colistin (antibiotic)– Nalidixic acid (antibiotic)– Sheep’s blood

(nutrients/hemolysis substrate)

• Used for:– Selective culture of Gram-

positive species– Distinguishing between

Gram-positives based on hemolysis

http://iws2.collin.edu/dcain/CCCCD%20Micro/columbiacnaagar.htm

E. faecalis E. aerogenes

E. coliS. xylosus

MacConkey Agar

• Selective• Differential• Special features:

– Crystal violet (dye)– Bile salts– Lactose (fermentation

substrate)– Neutral red (pH indicator)

• Used for:– Selective culture of Gram-

negative organisms– Distinguishing between Gram-

negatives based on lactose fermentationhttp://iws2.collin.edu/dcain/CCCCD%20Micro/macconkeyagar.htm

E. aerogenes E. coli

S. typhimurium S. epidermis

Eosin Methylene Blue (EMB) Agar

• Selective• Differential• Special features:

– Eosin (dye)– Methylene blue (dye)– Lactose (fermentation

substrate)

• Used for:– Selective culture of Gram-

negative species– Distinguishing between

Gram-negatives based on lactose fermentation

http://iws2.collin.edu/dcain/CCCCD%20Micro/embagar.htm

E. coli P. aeruginosa

S. xylosus E. aerogenes

Snyder Test Agar

• Selective• Special features:

– Acidic pH (4.8)– 2% glucose (fermentation

substrate)– Bromcresol green (pH

indicator)

• Used for:– Assessing levels of

Lactobacillus spp. organisms in the mouth

– Testing susceptibility to dental caries

http://iws2.collin.edu/dcain/CCCCD%20Micro/snydertest.htm

Temperature Classification

All Microbes Have an Optimal Growth Temperature

Figure 6.5

Psychrotrophs

Incubation at Multiple Temperatures Identifies Temperature Classification

Figure 6.4b

Examples of Temperature Classifications

Classification Characteristics Examples from Lab

Psychrophile Growth Range: -5 to 20 COptimum Temperature: ~10 C

Pseudomonas fluorescens*(*Actually a psychrotroph)

Mesophile Growth Range: 20 to 45 COptimum Temperature: ~37 C

Escherichia coli, Micrococcus luteus, Serratia marcescens, Saccharomyces

cerevisiae

Thermophile Growth Range: 35 to 80 COptimum Temperature: ~55 C Bacillus stearothermophilus

Hyperthermophile Growth Range: Above 80 COptimum Temperature: variable Archea such as Pyrolobus

http://iws2.collin.edu/dcain/CCCCD%20Micro/temperatureclassification.htm

Oxygen Classification

BHI Agar

1. Obligate aerobe2. Microaerophile3. Facultative anaerobe4. Aerotolerant anaerobe5. Obligate anaerobe

http://iws2.collin.edu/dcain/CCCCD%20Micro/bhitubes.htm

Sodium Thioglycollate Broth

1. Obligate anaerobe2. Obligate aerobe3. Aerotolerant anaerobe4. Facultative anaerobe

http://iws2.collin.edu/dcain/CCCCD%20Micro/thioglycollatebroth.htm

Control with Chemicals and Antibiotics

The Disk Diffusion Assay Measures Sensitivity

Figure 10.9

A Susceptibility Chart is Used to Determine Effects of Antibiotics