Notes

1
Notes

• MWA3 Due today
• MWA4 Due next week
• Gram Stain quiz next week
• Aseptic technique next week

2
TODAY- What we are doing

• Staining
• Differential staining continued
• Acid fast and Endospore
• Control organisms
• What are they and when do you use them?
• Aseptic Technique
• Discussion/demonstration technical difficulties
• Practice sterile transfers using tap H20
• You will be tested over your sterile technique
  next week (technical points)
• Environmental unknown
• Second step in purification process
• Check plates within 24-48 hours and Announce
  discuss expectations and open hours
• Gram stain quiz NEXT WEEK (20 pts)
• Announce discuss expectations

3
Gram Stain Quiz

• You will have 2-3 species in your unknown broth
• Bacillus megaterium - Gram , rod
• Escherichia coli - Gram -, rod
• Staphylococcus epidermidis - Gram , Cocci
• Spelling Counts!!!

4
Exercise 3 Part I

5
Differential Staining (cont.)

• Acid-Fast stain
• used to identify organisms of the genera
  Mycobacterium and Nocardia, including
  Mycobacterium tuberculosis
• contain waxes (mycolic acids) in their cell walls
  
• impervious to dyes such as those used in the Gram
  stain
• dyes can be driven into these organisms with
  heat
• Include a non-acid-fast control on the slide
  (e.g., Bacillus spp.)

6
Differential Staining What is a Control ?

• CONTROL ORGANISM An organism with a known
  reaction to a specific test that is used in
  comparative analysis.
• WHEN TO USE A CONTROL ORGANISM Always!

7
Acid-Fast Bacteria Cell Wall Structure
8
Acid-Fast MycobacteriumWhat they look like
Cluster of cells
Single cell
Mycobacterium avium (pink cells)
Mycobacterium tuberculosis (pink cells)
9
ACID-FAST STAIN procedure (p. 29-Photographic
Atlas)

• Clean Slide
• Prepare a dry mount of your organism
  Mycobacterium smegmatis with B. meg. for a
  control (heat fix).
• Place a piece of filter paper over the slide,
  hold it in place with a clothespin and saturate
  the paper with carbol fuschin.
• Gently heat the slide for 5 minutes. Add more
  carbol fuschin as the filter paper starts to dry
  out.
• Remove paper and gently rinse slide with
  distilled water.
• Decolorize slide with acid alcohol for 5-10
  seconds.
• Rinse with distilled water.
• Counterstain with methylene blue for 1 minute.
• Rinse with distilled water and blot dry.

10
Endospore Stain

• The second differential stain you will be
  performing today
• Endospore stain (Schaffer and Fulton staining
  method)
• The staining procedure is very similar to the
  acid-fast procedure.

11
Endospore Stain

• Endospore stain (Schaffer and Fulton)
• Some microorganisms (e.g., Bacillus megaterium)
  are able to form endospores. In contrast to
  vegetative cells, endospores are resistant to
  heat, dessication and chemicals, including
  stains.

12
Endospore Formation

• Bacteria that are resistent to environmental
  extremes frequently produce spores to allow
  survival during adverse conditions. The process
  of condensation of bacterial DNA within a series
  of membranes that acculumate calcium, dipicolinic
  acid and protein layers is known as sporulation.

IU School of Medicine, X525 Medical Microbiology
Mary Johnson, Ph.D.
13
Endospore Formation

• When an endospore-forming cell is stressed it
  encapsulates its DNA into a endospore
• If the cell remains stressed for an extended
  period of time, the outer cell wall breaks down
  leaving an exospore

14
Endospore Placement

• Placement can be
• terminal
• subterminal
• central
• Placement of the endospore is a taxonomic feature!

IU School of Medicine, X525 Medical Microbiology
Mary Johnson, Ph.D.
15
Endospore StainWhat they look like stained
Bacillus anthrasis spores
16
ENDOSPORE STAIN (p. 32 Photographic Atlas)

• Clean Slide.
• Prepare a dry mount of your organism Bacillus
  megaterium (heat fix).
• Place a piece of filter paper over the slide,
  hold it in place with a clothespin and saturate
  the paper with malachite green.
• Gently heat the slide for 5 minutes. Add more
  malachite green as the filter paper starts to dry
  out.
• Remove the paper and rinse the slide with water
  for 30 seconds.
• Counterstain with safranin stain for 20 seconds,
  then briefly rinse again with water.
• Blot dry and examine under 100X oil immersion
  spores will be green in pink cells.

17
Sterile Technique

• Aseptically transferring liquids by pipetting
  sterile media from one tube to another.
• This exercise requires (and teaches) dexterity
  and technical skill. In part 1 of exercise
  three, the student does not use sterile media for
  the transfers.
• In this portion of the exercise, it is necessary
  to stress the importance of manipulating the
  equipment required to perform sterile transfers
  even though the medium used is water.

18
Sterile Technique ProcedurePart I

• Transferring liquids (e.g., sterile broth or pure
  cultures) aseptically from one tube to another
  takes practice so that the liquid is not
  contaminated.
• When using sterile disposable pipettes, a Pipette
  Pump is attached to the top of the pipette in
  order to draw the liquid into the pipette. Never
  pipette by mouth.

19
Sterile Technique Procedure Part I

• Insert the pipette into the Pipette Pump and
  gently but firmly push in and twist (Fig. 3.1).
  Make certain that you do not contaminate the
  pipette by touching the tip with your hands.
• The blue Pipette Pump is for 1 ml pipettes the
  green one is for 5 and 10 ml pipettes.

20
Transfer to Broth Media in Bottles or Tubes

• a. After the lid is removed, flame the mouth of
  the bottle or tube.
• b. Holding the vessel at an angle, introduce the
  loop or needle into the liquid and agitate
  gently. If a pipette is used, a measured volume
  of liquid can be released using the pipettor.

21
Transfer to Broth Media in Bottles or Tubes

• c. Flame the mouth of the bottle or tube before
  returning the cover. Flame the loop or needle
  after use to incinerate any remaining organisms.
  Dispose of all contaminated pipettes or tips in
  the appropriate containers.

22
Sterile Technique Procedure Part I Begin

• Practice transferring water from a culture bottle
  to tubes aseptically in various amounts with the
  three different sizes of pipettes (1, 5 and 10
  ml).
• Next week you will use an enriched media to
  transfer and contamination will result in loss of
  technical points.

23
Begin Sterile Technique

• You should be able to accurately transfer volumes
  of 0.1, 0.5 and 1.0 ml using a 1.0 ml pipette
• 0.5, 1.0 and 5 ml with a 5.0 ml pipette
• 1.0, 5.0 and 10.0 ml with a 10.0 ml pipette.

24
Begin Sterile Technique

• Practice all these transfers until you are
  comfortable that you can transfer liquids
  aseptically (sterilely) and accurately (correct
  volume).
• Make sure to flame the lips of bottles and
  culture tubes before and after removing or adding
  liquids. In addition, do not place bottle caps
  or culture tube closures on your lab bench hold
  them in your hand.

25
Points for aseptic technique test (next week)

• 5 technique points are at stake
• Bottle contamination 1pt
• Bottle volume 1pt
• Tube contamination .25pt/tube (x6)
• Tube volume .25pt/tube (x6)

26
Environmental Isolate

• Inspect your attempt at purifying your
  environmental unknown. Your unknown should be
  purified at least three times.
• To check your culture make a wet mount and then
  two gram stains, one with only the environmental
  isolate and one with control organisms plus your
  environmental isolate.

27
Example of Gram Stain Quiz for Next Week

• Unknown
  Name
  
• 
  
  Date Sec
  
• Gram Stain Quiz
• Directions Perform a Gram Stain and fill out the
  blanks as you go. Be patient and stay calm, as
  you will be given ample time to practice and
  finish. If your technique doesnt work out the
  first time, clean another slide and start again.
  There are a minimum of 2 and a maximum of 3
  genera in each sample.
• Remember Spelling counts and THIS IS A QUIZ,
  therefore no talking or helping your neighbor.
  Raise your hand when you are finished and I will
  come to you. Good Luck!
• 1.The NUMBER of different genera in your unknown
  is, (circle the correct number).
• 1 2 3
• 2. The MORPHOLGY, GRAM STAIN, AND GRAM REACTION
  of the different genera are,
• Morphology Gram
  Stain Gram Reaction
• Cocci or Rod Color
  (pink or purple) G or G-
• A._________________ _________________ ____________
  ____
• B._________________ _________________ ____________
  ____
• C._________________ _________________ ____________
  ____
• 3. The names of the different genera are, (Genus
  and species).
• A.________________________ Please
  remember you have more than one chance at
• B.________________________ this quiz
  before you turn it in. Take your time and good
  luck.
• C.________________________