Review for Lab Practical _1 by linxiaoqin

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									Review for Lab Practical #1

          BIOL2421L
       Dr. Ann M. Davis
               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
         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
      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.
               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.
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
• Staphylococcus xylosus   • Escherichia coli
• Bacillus megaterium
                                                     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      – Culture of fungi (yeasts
                                                                    and molds)




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:
                                                             S. epidermis
                                                                               – 7.5% NaCl
                                                             S. aureus/
                                                             S. xylosus        – 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                                        • Used for:
        • Enriched                                               – Culture of fastidious organisms
        • Special features:                                      – Distinguishing between
                                                                   bacteria by hemolysis patterns
                – Sheep’s blood (nutrients,                        (esp. Streptococcus)
                  hemolysis substrate)




http://iws2.collin.edu/dcain/CCCCD%20Micro/hemolysis.htm
                                                Chocolate Agar
                                                                        • Enriched
                                                                        • Special features:
                                                                           – Boiled sheep’s blood
                                                                             (nutrients)
                                                                        • Used for:
http://iws2.collin.edu/dcain/CCCCD%20Micro/normalfloranasopharynx.htm
                                                                           – Growth of fastidious
                                                                             organisms (esp.
                                                                             Neisseria spp.)
                                                                           – Distinguish Neisseria
                                                                             spp. using oxidase test


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
E. faecalis                                         E. aerogenes   • Differential
                                                                   • Special features:
                                                                      – Colistin (antibiotic)
                                                                      – Nalidixic acid (antibiotic)
                                                                      – Sheep’s blood
                                                                        (nutrients/hemolysis
                                                                        substrate)
                                                                   • Used for:
    S. xylosus                                      E. coli           – Selective culture of Gram-
                                                                        positive species
                                                                      – Distinguishing between
                                                                        Gram-positives based on
                                                                        hemolysis
http://iws2.collin.edu/dcain/CCCCD%20Micro/columbiacnaagar.htm
                                         MacConkey Agar
                                                                • Selective
E. aerogenes                                          E. coli
                                                                • Differential
                                                                • Special features:
                                                                   – Crystal violet (dye)
                                                                   – Bile salts
                                                                   – Lactose (fermentation
                                                                     substrate)
                                                                   – Neutral red (pH indicator)
                                                                • Used for:
                                                                   – Selective culture of Gram-
S. typhimurium                                  S. epidermis         negative organisms
                                                                   – Distinguishing between Gram-
                                                                     negatives based on lactose
http://iws2.collin.edu/dcain/CCCCD%20Micro/macconkeyagar.htm
                                                                     fermentation
           Eosin Methylene Blue (EMB) Agar
                                                                  • Selective
     E. coli                                      P. aeruginosa   • Differential
                                                                  • Special features:
                                                                     – Eosin (dye)
                                                                     – Methylene blue (dye)
                                                                     – Lactose (fermentation
                                                                       substrate)
                                                                  • Used for:
                                                                     – Selective culture of Gram-
                                                                       negative species
     S. xylosus                                   E. aerogenes       – Distinguishing between
                                                                       Gram-negatives based on
                                                                       lactose fermentation
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
     Classification                            Characteristics                     Examples from Lab

                                    Growth Range: -5 to 20 C                   Pseudomonas fluorescens*
      Psychrophile
                                   Optimum Temperature: ~10 C                  (*Actually a psychrotroph)


                                                                           Escherichia coli, Micrococcus luteus,
                                    Growth Range: 20 to 45 C
       Mesophile                                                           Serratia marcescens, Saccharomyces
                                   Optimum Temperature: ~37 C
                                                                                        cerevisiae


                                    Growth Range: 35 to 80 C
     Thermophile                                                               Bacillus stearothermophilus
                                  Optimum Temperature: ~55 C


                                   Growth Range: Above 80 C
 Hyperthermophile                                                               Archea such as Pyrolobus
                                 Optimum Temperature: variable

http://iws2.collin.edu/dcain/CCCCD%20Micro/temperatureclassification.htm
Oxygen Classification
                     BHI Agar


1.   Obligate aerobe
2.   Microaerophile
3.   Facultative anaerobe
4.   Aerotolerant anaerobe
5.   Obligate anaerobe



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


1.   Obligate anaerobe
2.   Obligate aerobe
3.   Aerotolerant anaerobe
4.   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
            For More Review…
• Dr. Carroll Bottoms’s Website:
  http://iws2.collin.edu/cbottoms/microbiology
  _laboratory_schedule.htm
  – Chart for Practical I
  – Review for Practical
• Microbiology Tutorial Website:
  http://iws2.collin.edu/dcain/CCCCD%20Micro
  /tutorial.htm

								
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