LAB PRACTICAL I
Lab Exercise 1, 2-1, 2-2, 2-3, 2-4, 2-13,1-4, 1-5, 3-1, 3-6, 3-7, 3-9, 3-10, 6-2, 7-2
Note: Study all the learning objectives given for the following labs. Go to each week folder and
study the learning objectives. Study ppt. 3 and watch recorded lectures.
Lab 1: Safety and Laboratory Guidelines (page 1)
Exercise 1-4 Common aseptic Transfers and inoculation methods. Page 31
1. Define culture, pure culture, aseptic technique.
2. Learn the steps to carry out aseptic transfer from a broth pure culture to sterile Broth
tube. Page 35. (You will do a virtual simulation lab to practice, please look at the graded
folder)
3. Explain the aseptic transfer from a nutrient agar slant to a sterile nutrient agar slant.
Page 38 (You will do a virtual simulation lab to practice, please look at the graded folder)
4. Explain the aseptic transfer from nutrient agar slant culture to sterile broth. Page 39
(You will do a virtual simulation lab to practice, please look at the graded folder)
Exercise 1-5 Streak Plate Methods of Isolation. Page 45
1. What is streak plate method?
2. What is the purpose of streak plate?
3. What are types of streak plate?
4. Demonstrate streak plate on nutrient agar using aseptic technique.
Exercise 2-1 Ubiquity of Microorganisms. Page 61.
Exercise 2-2 Colony morphology. Page 67
1. You will view colony characteristics and identify whole colony shape, margin, and
elevation of colony on nutrient agar plate. (Study page 68 to 76 in lab manual, watch
video)
Exercise 2-3 Growth patterns on slant. Page 79
1. Define an agar slant
2. What is the purpose of agar slant.
3. Explain type of growth, texture and margin. (Study page # 79 from lab manual)
Exercise 2-4 Growth patterns in broth. Page 83
1. Identify pellicle, sediment, uniform fine turbidity and flocculent type of growth in nutrient
broth. (Please see the images in your lab manual, page 83)
Exercise: 3-1 to 3-3: Microscopy Page 141
1. Define and describe parts of Bright field Compound Microscope.
2. List different types of Light Microscope.
3. Define, resolution, numerical aperture, working distance, contrast, magnification power.
4. Learn proper technique to use microscope.
5. Practice visualizing bacteria and demonstrate successful focusing to get a sharp image
under different magnification.
Exercise 3-4 Simple stains. Page 177
Exercise 3-6 Gram Stain page 69
Exercise 3-7 Acid fast stain Page 195
Exercise 3-9 Endospore stain Page 205
Exercise 3-10: Bacterial motility: wet mount and hanging drop preparations. Page 211
1. Demonstrate making a wet mount slide of E.coli by hanging drop method.
2.
3.
4.
5.
What are the advantage of hanging drop method for observing motility?
Focus under 100 x objective and visualize motility.
How bacteria move?
Explain the difference between true motility and Brownian motion
Review for Lab Practical I
Lab 1: Safety and Laboratory Guidelines (page 1)
1) Biosafety levels - You should be able to read the points of BSL, and be able to recognize
the number of biosafety levels. Remember some examples under each category.
● BSL 1 - Low risk, unlikely to cause disease (Ex. E. coli)
● BSL 2 - Moderate risk, containment, diseases of varying severity (Ex. Influenza, HIV,
Lyme disease)
● BSL 3 - High risk, serious/potentially lethal (Ex. Tuberculosis)
● BSL 4 - Very high risk, life-threatening (Ex. Ebola)
2) Disposal followed in our lab
3) Identifying lab safety equipment
Exercise: 3-1 to 3-3: Microscopy Page 141
1) Parts of compound microscope
2) Resolution
3) Common shapes of bacteria
4)
5)
6)
7)
Gram positive and Gram negative bacteria (Know the one we work in our lab)
Know the name of some pathogenic bacteria - viruses, bacteria, fungi, and parasites
Advantage of oil immersion oil
Name some eukaryotic pathogens.
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Candida.
Cryptococcus.
Plasmodium.
Giardia.
Candida.
Cryptococcus.
Plasmodium.
Giardia.
8) You should be able to calculate total magnification
9) Define contrast, working distance, field of view
Exercise 3-10: Bacterial motility: wet mount and hanging drop preparations.
1. Describe the steps of hanging-drop procedure to view motility.
2. What is the difference between true motility and Brownian motion?
In true motility, bacteria change their position with respect to the original
position, whereas in Brownian movement it will not change its position (constant
vibratory movement)
3. What type of structure bacteria use to move?
Flagella
4. Why is Vaseline added?
Vaseline-sealed depression also slows down the drying-out process, so the
organisms can be observed for longer periods.
Exercise 2-2 Colony morphology. Page 67
Note: Be familiar with different media such as nutrient agar, nutrient broth, agar slant and
agar deep.
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Use appropriate term to describe colony appearance, margin and elevation
Exercise 2-3 Growth patterns on slant. Page 79
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Use appropriate term to describe the colony appearance and texture
·
What is the application of slant?
used to culture bacterial cells for identification. Attempting to identify bacteria from a
large sample is difficult because bacteria are small and can be difficult to find
Exercise 2-4 Growth patterns in broth. Page 83
1) Use appropriate terms to describe bacterial growth in broth.
Exercise 1-4 Common aseptic Transfers and inoculation methods. Page 31
1) Describe the pattern of growth on agar slants.
2) Differentiate a motile from non-motile bacteria in a deep.
Motile bacteria will grow out from the stab line while non-motile bacteria are present
only along the stab line.
Exercise 1-4 Common aseptic Transfers and inoculation methods. Page 31
1) Describe the steps of aseptic technique in inoculating procedures. (open-ended
question)
Exercise 1-5 Streak Plate Methods of Isolation. Page 45
1) What is the significance of streak-plate method?
or isolation is to produce isolated colonies of an organism on an agar plate. This is
useful when you need to separate organisms in a mixed culture or when you need to
study the colony morphology of an organism.
2) Be familiar with proper technique of streaking a plate.
3) What type of streak plate we did in our lab?
- Basic streak, T-streak, and 4 Quadrant streak
Exercise 3-4 Simple stains. Page 177
1) Know the function of stain, and types of stains and their application.
2) Name some basic and acid stains.
Table 2. Simple Stains
Stain Type
Specific Dyes
Basic stains
Methylene blue, crystal violet, malachite green,
basic fuschsin, carbolfuschsin, safranin
Acidic stains
Eosine, acid fuchsin, rose bengal, Congo red
Exercise 3-6 Gram Stain page 69
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Describe the steps of gram stain
3) Know the primary stain, mordant, decolorizer, and counter stain.
4) Name gram positive and negative bacteria.
Gram-positive: Staphylococcus aureus, Staphylococcus epidermidis, and
cereus
Bacillus
Gram-negative: E. coli, Serratia marcescens, and Meningococcus
5) Why an old culture can give false negative results.
Exercise 3-7 Acid fast stain Page 195
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Know the primary stain, mordant, decolorizer, and counter stain.
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Name acid-fast bacteria genus - Mycobacterium and Nocardia
List the steps of acid-fast staining.
Exercise 3-9 Endospore stain Page 205
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Know the primary stain, mordant, decolorizer, and counter stain.
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Name a bacteria that makes endospores - Bacillus cereus
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Describe the steps of endospore staining
Exercise 6-2 Standard plate count (viable count) page 411
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What is a countable plate. - measure colony forming units of bacteria which are in
range between 30 and 300 colonies.
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Calculate Total and current dilution. - sample/(diluent + sample)
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For example: If 1ml of culture is diluted with 9ml of sterile dilutant, what is the total dilution
of the culture? - sample/(diluent + sample) (1/(9+1) = 1/10 or 1:10
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What is the total dilution of 6 th dilution series.
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Know how to calculate cfu/ml
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Cfu/ml= # Of CFUX D/V - (no. of colonies x dilution factor) / volume of culture plate