Review for Lab Practical #1 MLAB 352 Dr. 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 (primary stain) (mordant) (decolorizer) (counterstain) http://iws2.collin.edu/dcain/CCCCD%20Micro/gram_stain.htm http://student.ccbcmd.edu/courses/bio141/lecguide/unit1/prostruct/grmix.html

Examples of Gram-Positive and Gram-Negative Organisms Staphylococcus xylosus Bacillus megaterium Escherichia coli

(decolorizer/counterstain) Capsule Stain NO HEAT FIXATION! (primary stain) (decolorizer/counterstain) Alcaligenes faecalis Enterobacter aerogenes Klebsiella pneumoniae Serratia marcescens http://iws2.collin.edu/dcain/CCCCD%20Micro/capsule_stain.htm

Acid-Fast Stain Mycobacterium smegmatis (primary stain) (decolorizer) (counterstain) Mycobacterium smegmatis Figure 4.18 http://iws2.collin.edu/dcain/CCCCD%20Micro/acid_fast.htm

Endospore Stain Bacillus megaterium (primary stain) (decolorizer) (counterstain) Bacillus megaterium http://iws2.collin.edu/dcain/CCCCD%20Micro/endospore.htm

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: Used for: None Culture of non-fastidious organisms http://commons.wikimedia.org/wiki/File:Agar_Plate.jpg

Sabouraud Agar/Broth Selective media Special features: Used for: 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. S. epidermis S. aureus/ S. xylosus http://iws2.collin.edu/dcain/CCCCD%20Micro/normalskinflora.htm

Blood Agar Differential Enriched Special features: Used for: 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: Used for: 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 E. faecalis E. aerogenes S. xylosus E. coli http://iws2.collin.edu/dcain/CCCCD%20Micro/columbiacnaagar.htm

MacConkey Agar Selective Differential Special features: Used for: 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 fermentation E. aerogenes E. coli S. typhimurium S. epidermis http://iws2.collin.edu/dcain/CCCCD%20Micro/macconkeyagar.htm

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 E. coli P. aeruginosa S. xylosus E. aerogenes http://iws2.collin.edu/dcain/CCCCD%20Micro/embagar.htm

Snyder Test Agar Selective Special features: Used for: 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 Psychrotrophs Figure 6.5

Incubation at Multiple Temperatures Identifies Temperature Classification Figure 6.4b

Examples of Temperature Classifications Characteristics Examples from Lab Psychrophile Growth Range:  -5 to 20 C Optimum Temperature: ~10 C Pseudomonas fluorescens* (*Actually a psychrotroph) Mesophile Growth Range: 20 to 45 C Optimum Temperature: ~37 C Escherichia coli, Micrococcus luteus, Serratia marcescens, Saccharomyces cerevisiae Thermophile Growth Range: 35 to 80 C Optimum Temperature:  ~55 C Bacillus stearothermophilus Hyperthermophile Growth Range: Above 80 C Optimum Temperature: variable Archea such as Pyrolobus http://iws2.collin.edu/dcain/CCCCD%20Micro/temperatureclassification.htm

Oxygen Classification

BHI Agar Obligate aerobe Microaerophile Facultative anaerobe Aerotolerant anaerobe Obligate anaerobe http://iws2.collin.edu/dcain/CCCCD%20Micro/bhitubes.htm

Sodium Thioglycollate Broth Obligate anaerobe Obligate aerobe Aerotolerant anaerobe 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