Review for lab practical 1
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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.

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Review for Lab Practical #1

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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

GRAM-POSITIVE

GRAM-NEGATIVE

Escherichia coli

  • Staphylococcus xylosus

  • Bacillus megaterium


Capsule Stain

NO HEAT FIXATION!

  • Alcaligenes faecalis

  • Enterobacter aerogenes

  • Klebsiella pneumoniae

  • Serratia marcescens

(primary stain)

(decolorizer/counterstain)

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


Acid-Fast Stain

(primary stain)

(decolorizer)

(counterstain)

  • Mycobacterium smegmatis

Figure 4.18

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


Endospore Stain

(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:

    • 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.

S. epidermis

S. aureus/

S. xylosus

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


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

E. aerogenes

E. faecalis

E. coli

S. xylosus

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


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 fermentation

E. aerogenes

E. coli

S. epidermis

S. typhimurium

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

P. aeruginosa

E. coli

S. xylosus

E. aerogenes

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


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

Psychrotrophs

Figure 6.5


Incubation at Multiple Temperatures Identifies Temperature Classification

Figure 6.4b


Examples of Temperature Classifications

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


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