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

Review for Lab Practical #1

MLAB 352

Dr. Mohamed A. El-Sakhawy


Lecture outline

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

Format of the Lab Practical


Biosafety

Biosafety


General biosafety rules

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

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

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

Staining


Types of bacterial stains

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

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

A Smear is Prepared by Spreading Bacteria on a Glass Slide

Figure 4.15


Heat fixing a bacterial smear

Heat-Fixing a Bacterial Smear

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


Gram stain

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

Examples of Gram-Positive and Gram-Negative Organisms

GRAM-POSITIVE

GRAM-NEGATIVE

Escherichia coli

  • Staphylococcus xylosus

  • Bacillus megaterium


Capsule stain

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

Acid-Fast Stain

(primary stain)

(decolorizer)

(counterstain)

  • Mycobacterium smegmatis

Figure 4.18

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


Endospore stain

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


Four way plate streak1

Four-Way Plate Streak

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


Plate counts

Plate Counts


Viable plate counts from serial dilutions

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

Calculation of CFU/ml for Urine Specimens

  • Count colonies

    • CFU/ml = # colonies/calibrated loop volume

  • Threshold: 1 x 105 CFU/ml


Media types

Media Types


Nutrient agar na

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

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

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

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

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

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

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

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

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

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

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

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

Temperature Classification


All microbes have an optimal growth temperature

All Microbes Have an Optimal Growth Temperature

Psychrotrophs

Figure 6.5


Incubation at multiple temperatures identifies temperature classification

Incubation at Multiple Temperatures Identifies Temperature Classification

Figure 6.4b


Examples of temperature classifications

Examples of Temperature Classifications

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


Oxygen classification

Oxygen Classification


Bhi agar

BHI Agar

  • Obligate aerobe

  • Microaerophile

  • Facultative anaerobe

  • Aerotolerant anaerobe

  • Obligate anaerobe

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


Sodium thioglycollate broth1

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

Control with Chemicals and Antibiotics


The disk diffusion assay measures sensitivity

The Disk Diffusion Assay Measures Sensitivity

Figure 10.9


A susceptibility chart is used to determine effects of antibiotics

A Susceptibility Chart is Used to Determine Effects of Antibiotics


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