Class Four

1
Class Four

• Microbiology
• 2006

2
Quiz

• Quiz Number One Open Response and
  Identification
• Microscope
• Aseptic techniques
• Culture transfer techniques
• Types of Media
• Safety consideration

3
Case Study

• Read Case Study One
• Answer the questions on the case study for class
  five
• Use the links on your web page to research
• Answer questions in complete sentences
  preferred format answers done on the web
  processor

4
Culture Results

• Observe your eight cultures
• Four gram positive
• Four gram negative
• Record results in the table for all organisms
• Record results of colony characteristics and
  growth

5
Part Two - Staining

• How to Make a Smear
• Stain with Gram Stain
• Stain with Simple Stain

6
How to Make a Smear

• 1.  Label the frosted side of your slide with
  your initials, the name of the organism, and the
  date.  On this side draw a circle in the clear
  section of the slide.
• 2.  Turn the slide over.  You will make your
  smear on this side.
• If you are using broth follow these
  directions
• Flame your inoculating loop.
• Use aseptic technique and remove the top of the
  culture tube, flame the mouth or the culture
  tube, and dip the loop into the broth.  Make sure
  that the loop is filled.
• Transfer the loopful of broth and bacteria to the
  slide. Using a circular motion, spread the broth
  on the slide.
• This is now a " smear"
• Allow the smear to dry
• When the smear has been allowed to " air dry" ,
  pass the smear through the flame to " heat fix" -
  Heat fixation causes the proteins and cell parts
  to coagulate and stick to the slide.
• Let the slide cool.

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How to Make a Smear

• If you are using colonies from a plate(
  Petri Dish)
• Place a drop of water on the slide.
• Using aseptic technique, " pick" a colony from
  the Plate that you want to Gram Stain.
• Using a circular motion spread move the
  inoculating loop in a circular motion in the drop
  of water. Start in the center of the drop and
  move in a circular motion to the outside of the
  drop.  The objective is to have fewer cells on
  the outside of the circle. 
• Follow the directions from above
• Don't forget to " air dry" and to " heat
  fix" before staining

8
The Gram Stain

• Gram Stain- See Gram Stain Directions on separate
  page.  Please refer to pages 71-73 in your
  laboratory manual.
•   
• All staining work is to be done at the sink
• Care should be taken to work directly over the
  sink
• Place drop(s) of crystal violet stain on the
  smear ( 1 minute)
• Rock or roll the slide to cover the area
• Use the water bottle to drip water down the slide
  
• Place drop(s) of iodine on the slide ( 1 minute)
• Place drops of alcohol on the slide 10 seconds (
  KEY do not leave on longer than 10 seconds or
  it will decolorize)
• Place drop(s) of saffranin on the slide for 1
  minute
• Rinse with water from the bottle
• Let the slide air dry

9
Simple Stain

• Simple Stains- Crystal violet and methylene blue
• 1.  Place a drop(s) of stain over the smear. Make
  sure it covers the entire area of the smear. 
  Leave the stain on the smear for one minute. 
  Rinse with water from the bottle. 
• 2. Refer to page 64 for cellular morphology.

10
Typical Gram Stain

• http//www.uphs.upenn.edu/bugdrug/antibiotic_manua
  l/Gram3.htm
• http//www.uphs.upenn.edu/bugdrug/antibiotic_manua
  l/Gramstains/small/tocframeset1.htm

11
Staphylococcus
12
Gram Negative bacteria
13
Bacterial Cell Morphology
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