Overview

1
Overview

• Turn in MWA 2 and pre-lab
• Discuss MWA 3
• Gram stain quiz is next week
• Env. Isolate
• Staining
• Sterile Technique Part I

2
Review

• So far we have used both phase contrast (for
  live, unstained organisms) and brightfield (for
  stained organisms) microscopy
• Last week we did
• the simple stain (purpose to get an idea of
  morphology),
• the Gram stain (to differentiate between types of
  organisms)
• capsule stain (to look at the capsules some
  organisms produce-this is an example of a
  differential stain)

3
Review

• Staining an organism helps increase the contrast
• Remember the iris diaphragm- helps adjust
  contrast in brightfield microscopy (does not work
  in phase contrast)

4
Environmental Isolate

• Last week you should have gotten a good idea for
  how fast it grows-if not, make sure to re-streak
  and check this week
• Check your plates for purity
• Re-streak from section 3, we will do a wet mount
  next week

5
Sterile Technique ProcedurePart I

• The goal of using sterile technique is to
  transfer a substance from one area to another
  without contaminating the original container, the
  sample, or the receiving container
• In order to achieve this goal, you must be very
  careful with your technique
• Today we will be practicing with water and using
  pipettes

6
Sterile Technique

• Set up everything you will need at your bench
• Open your pipette by using the tabs at the top of
  the package-DO NOT take your pipette all the way
  out of the plastic yet
• Attach your pipette pump to the top of your
  pipette

7
Sterile Technique

• Without setting your pipette down, pick up the
  tube you will take liquid from
• Make sure the lid is loose enough you can open it
  easily
• Remove your pipette from the plastic
• Uncap your tube

8
Sterile Technique

• Flame the lip of your tube/bottle, do not set cap
  down
• Insert your pipette and draw out the desired
  amount of liquid
• Flame the lip of the tube, re-cap, and pick up
  your next tube without setting down your pipette
• Uncap, flame, and transfer the liquid

9
Sterile Technique

• Flame the lip, recap and set down both
  tubes/bottles
• Take your pipette and replace it in the plastic
  wrap (without lighting it on fire or touching the
  pipette to anything)
• Place your used pipette in the Nalgene buckets on
  the lab bench

10
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.

11
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 is the test. You will use an enriched
  media to transfer and contamination will count
  against you.

12
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.

13
Sterile Technique

• Sterile (Aseptic) Technique (Part 1) Page 3-2 in
  the Lab Manual.
• 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.

14
Differential Staining (cont.)

• Acid-Fast stain
• used to identify organisms of the genera
  Mycobacterium and Nocardia, including
  Mycobacterium tuberculosis
• Mycobacterium contain waxes (mycolic acids,
  http//www.cyberlipid.org/fa/acid0006.htm) 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.)

15
Differential Staining What is a Control ?

• CONTROL ORGANISM An organism with a known
  reaction to a specific test that is used in
  comparative analysis
• Essentially its a way to check your procedure
  and materials

16
Acid-Fast Bacteria Cell Wall Structure
17
Acid-Fast MycobacteriumWhat they look like
Cluster of cells
Single cell
Mycobacterium avium (pink cells)
Mycobacterium tuberculosis (pink cells)
18
ACID-FAST STAIN procedure (p. 29-Photographic
Atlas)

• Clean Slide
• Prepare a dry mount of your organism
  Mycobacterium smegmatis (heat fix).
• Place several wet paper towels around the Bunsen
  burner.
• 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.

19
Endospore Stain

• Endospore stain (Schaffer and Fulton staining
  method)
• The staining procedure is very similar to the
  acid-fast procedure

20
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.

21
Endospore Formation
22
Endospore Placement

• Placement can be
• terminal
• subterminal
• central

Pictures from Brock Biology of Microorganisms
23
Endospore StainWhat they look like stained
A. Vegetative Cell B. Endospore
24
ENDOSPORE STAIN (p. 32 Photographic Atlas)

• Clean Slide.
• Prepare a dry mount of your organism Bacillus
  megaterium (heat fix).
• Place a wet paper towel around the Bunsen burner.
• 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.

25
Example of Gram Stain TEST for Next Week

• Unknown
  Name
  
• 
  
  Date Sec
  
• Gram Stain TEST
• 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
• B.

26
What is this?
http//slph.state.nc.us/public2/images/Yersinia_pe
stis_microscopic.jpg
27
How about this one?
http//medlib.med.utah.edu/kw/derm/mml/24850028.jp
g
28
and this one?
http//www.wzw.tum.de/micbio/cms/UserFiles/Image/n
euhaus3.jpg