Review for Lab Practical 1

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Review for Lab Practical 1

• BIOL2421L
• Dr. Ann M. Davis

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Lecture Outline

• Exam format
• Biosafety
• Staining
• Four-way plate streak
• Plate counts
• Media types
• Temperature classification
• Oxygen classification
• Control with chemicals and antibiotics

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Format of the Lab Practical
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General Exam Format

• 24 stations, 80-90 questions
• One station per desk
• 1-5 questions per station
• 1-2 bonus questions
• 3 minutes per station
• Entire class rotates as a group
• YOU MAY NOT MOVE OUT OF TURN!
• 15 minutes at the end to go back
• Answer sheet will be provided for you

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Requirements for Answers

• Spelling must be correct.
• Organism names must be completely spelled out.
• Staphylococcus aureus ? full credit
• S. aureus ? half credit
• Names of reagents and media components must be
  complete.
• Crystal violet ? full credit
• Violet ? half credit
• Numerical answers
• Include the units.
• Correctly express with scientific notation when
  appropriate to do so.

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Rules of the Exam

• NO notes, notecards, study guides, books, or
  other aids.
• Bring only a pencil.
• NO cell phones.
• Turn off and stow in your bag.
• YOU MAY NOT WORK TOGETHER ON ANY PART OF THIS
  EXAM!
• DO NOT discuss the exam outside of class until
  given permission to do so by the instructor.
• VIOLATION OF THE CODE OF ACADEMIC ETHICS WILL
  RESULT IN NO CREDIT.

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Biosafety
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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.

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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.

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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.

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Staining
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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

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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

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A Smear is Prepared by Spreading Bacteria on a
Glass Slide
Figure 4.15
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Heat-Fixing a Bacterial Smear
http//iws2.collin.edu/dcain/CCCCD20Micro/Heat_fi
x.htm
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Gram Stain
(primary stain)
(mordant)
(decolorizer)
(counterstain)
http//iws2.collin.edu/dcain/CCCCD20Micro/gram_st
ain.htm
http//student.ccbcmd.edu/courses/bio141/lecguide/
unit1/prostruct/grmix.html
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Examples of Gram-Positive and Gram-Negative
Organisms

• GRAM-POSITIVE

• GRAM-NEGATIVE

• Staphylococcus xylosus
• Bacillus megaterium

• Escherichia coli

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Capsule Stain
NO HEAT FIXATION!
(primary stain)
(decolorizer/counterstain)

• Alcaligenes faecalis
• Enterobacter aerogenes
• Klebsiella pneumoniae
• Serratia marcescens

http//iws2.collin.edu/dcain/CCCCD20Micro/capsule
_stain.htm
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Acid-Fast Stain
(primary stain)
(decolorizer)
(counterstain)

• Mycobacterium smegmatis

Figure 4.18
http//iws2.collin.edu/dcain/CCCCD20Micro/acid_fa
st.htm
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Endospore Stain
(primary stain)
(decolorizer)
(counterstain)

• Bacillus megaterium

http//iws2.collin.edu/dcain/CCCCD20Micro/endospo
re.htm
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Four-Way Plate Streak
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Four-Way Plate Streak
http//iws2.collin.edu/dcain/4way.htm
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Plate Counts
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Viable Plate Counts from Serial Dilutions

• TNTC Too Numerous To Count
• More than 300 colonies
• TFTC Too Few To Count
• Less than 30 colonies

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Calculation of CFU/ml for Urine Specimens

• Count colonies
• CFU/ml colonies/calibrated loop volume
• Threshold 1 x 105 CFU/ml

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Media Types
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Nutrient Agar (NA)

• Special features
• None
• Used for
• Culture of non-fastidious organisms

http//commons.wikimedia.org/wiki/FileAgar_Plate.
jpg
28
Sabouraud Agar/Broth

• Selective media
• Special features
• Acidic pH (5.5)
• Used for
• Culture of fungi (yeasts and molds)

http//iws2.collin.edu/dcain/CCCCD20Micro/saboura
udagar.htm
http//www.jlindquist.net/generalmicro/dfnewgfbpag
e.html
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Brain Heart Infusion (BHI) Agar

• Special features
• None
• Used for
• BHI agar deeps used to determine oxygen
  classification

http//iws2.collin.edu/dcain/CCCCD20Micro/bhitube
s.htm
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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/CCCCD20Micro/thiogly
collatebroth.htm
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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/CCCCD20Micro/normals
kinflora.htm
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Blood Agar

• Differential
• Enriched
• Special features
• Sheeps blood (nutrients, hemolysis substrate)

• Used for
• Culture of fastidious organisms
• Distinguishing between bacteria by hemolysis
  patterns (esp. Streptococcus)

http//iws2.collin.edu/dcain/CCCCD20Micro/hemolys
is.htm
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Chocolate Agar

• Enriched
• Special features
• Boiled sheeps blood (nutrients)
• Used for
• Growth of fastidious organisms (esp. Neisseria
  spp.)
• Distinguish Neisseria spp. using oxidase test

http//iws2.collin.edu/dcain/CCCCD20Micro/normalf
loranasopharynx.htm
http//www.medschool.lsuhsc.edu/microbiology/DMIP/
bsoxid.jpg
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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/CCCCD20Micro/normals
kinflora.htm
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Columbia C-NA (CCNA) Agar

• Selective
• Differential
• Special features
• Colistin (antibiotic)
• Nalidixic acid (antibiotic)
• Sheeps blood (nutrients/hemolysis substrate)
• Used for
• Selective culture of Gram-positive species
• Distinguishing between Gram-positives based on
  hemolysis

E. faecalis
E. aerogenes
E. coli
S. xylosus
http//iws2.collin.edu/dcain/CCCCD20Micro/columbi
acnaagar.htm
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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. typhimurium
S. epidermis
http//iws2.collin.edu/dcain/CCCCD20Micro/macconk
eyagar.htm
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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/CCCCD20Micro/embagar
.htm
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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/CCCCD20Micro/snydert
est.htm
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Temperature Classification
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All Microbes Have an Optimal Growth Temperature
Psychrotrophs
Figure 6.5
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Incubation at Multiple Temperatures Identifies
Temperature Classification
Figure 6.4b
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Examples of Temperature Classifications
http//iws2.collin.edu/dcain/CCCCD20Micro/tempera
tureclassification.htm
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Oxygen Classification
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BHI Agar

• Obligate aerobe
• Microaerophile
• Facultative anaerobe
• Aerotolerant anaerobe
• Obligate anaerobe

http//iws2.collin.edu/dcain/CCCCD20Micro/bhitube
s.htm
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Sodium Thioglycollate Broth

• Obligate anaerobe
• Obligate aerobe
• Aerotolerant anaerobe
• Facultative anaerobe

http//iws2.collin.edu/dcain/CCCCD20Micro/thiogly
collatebroth.htm
46
Control with Chemicals and Antibiotics
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The Disk Diffusion Assay Measures Sensitivity
Figure 10.9
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A Susceptibility Chart is Used to Determine
Effects of Antibiotics
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For More Review

• Dr. Carroll Bottomss Website
  http//iws2.collin.edu/cbottoms/microbiology_labor
  atory_schedule.htm
• Chart for Practical I
• Review for Practical
• Microbiology Tutorial Website
  http//iws2.collin.edu/dcain/CCCCD20Micro/tutoria
  l.htm