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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

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
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.
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
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 streak1
Four-Way Plate Streak

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

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
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

examples of temperature classifications
Examples of Temperature Classifications

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

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

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