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
• Staphylococcus xylosus
• Bacillus megaterium
GRAM-NEGATIVE
• Escherichia coli
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
(primary stain)
(decolorizer)
(counterstain)
• Mycobacterium smegmatis
http://iws2.collin.edu/dcain/CCCCD%20Micro/acid_fast.htm
Figure 4.18
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:
http://iws2.collin.edu/dcain/CCCCD%20Micro/sabouraudagar.htm
http://www.jlindquist.net/generalmicro/dfnewgfbpage.html
– Culture of fungi (yeasts
and molds)
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:
S. epidermis
S. aureus/
S. xylosus
– 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
Blood Agar
• Differential
• Enriched
• Special features:
• Used for:
– Sheep’s blood (nutrients,
hemolysis substrate)
http://iws2.collin.edu/dcain/CCCCD%20Micro/hemolysis.htm
– Culture of fastidious organisms
– Distinguishing between
bacteria by hemolysis patterns
(esp. Streptococcus)
Chocolate Agar
• Enriched
• Special features:
– Boiled sheep’s blood
(nutrients)
• Used for:
http://iws2.collin.edu/dcain/CCCCD%20Micro/normalfloranasopharynx.htm
http://www.medschool.lsuhsc.edu/microbiology/DMIP/bsoxid.jpg
– Growth of fastidious
organisms (esp.
Neisseria spp.)
– Distinguish Neisseria
spp. using oxidase test
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
E. faecalis
E. aerogenes
• Selective
• Differential
• Special features:
– Colistin (antibiotic)
– Nalidixic acid (antibiotic)
– Sheep’s blood
(nutrients/hemolysis
substrate)
• Used for:
S. xylosus
E. coli
http://iws2.collin.edu/dcain/CCCCD%20Micro/columbiacnaagar.htm
– Selective culture of Grampositive species
– Distinguishing between
Gram-positives based on
hemolysis
MacConkey Agar
E. aerogenes
E. coli
• Selective
• Differential
• Special features:
– Crystal violet (dye)
– Bile salts
– Lactose (fermentation
substrate)
– Neutral red (pH indicator)
• Used for:
S. typhimurium
S. epidermis
http://iws2.collin.edu/dcain/CCCCD%20Micro/macconkeyagar.htm
– Selective culture of Gramnegative organisms
– Distinguishing between Gramnegatives based on lactose
fermentation
Eosin Methylene Blue (EMB) Agar
E. coli
P. aeruginosa
• Selective
• Differential
• Special features:
– Eosin (dye)
– Methylene blue (dye)
– Lactose (fermentation
substrate)
• Used for:
S. xylosus
E. aerogenes
http://iws2.collin.edu/dcain/CCCCD%20Micro/embagar.htm
– Selective culture of Gramnegative species
– Distinguishing between
Gram-negatives based on
lactose fermentation
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
Classification
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
1.
2.
3.
4.
5.
Obligate aerobe
Microaerophile
Facultative anaerobe
Aerotolerant anaerobe
Obligate anaerobe
http://iws2.collin.edu/dcain/CCCCD%20Micro/bhitubes.htm
Sodium Thioglycollate Broth
1.
2.
3.
4.
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