Transcript Class Four - Welcome to Cherokee High School

Class Four
Microbiology
2006
Quiz
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Quiz Number One – Open Response and
Identification
Microscope
Aseptic techniques
Culture transfer techniques
Types of Media
Safety consideration
Case Study
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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
Culture Results
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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
Part Two - Staining
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How to Make a Smear
Stain with Gram Stain
Stain with Simple Stain
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
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*****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
The Gram Stain
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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
Simple Stain
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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.
Typical Gram Stain
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http://www.uphs.upenn.edu/bugdrug/an
tibiotic_manual/Gram3.htm
http://www.uphs.upenn.edu/bugdrug/an
tibiotic_manual/Gramstains/small/tocf
rameset1.htm
Staphylococcus
Gram Negative bacteria
Bacterial Cell Morphology