BIO 150 Spring 2022
Microbiology Lab Exam 1 Review Topics
There will be 40 stations, worth 2 points each (+ 2 extra-credit questions). You will be observing plates,
tubes and microscope slides, and answering questions (multiple-choice) about them. You will also have
questions on safety, technique, and procedures.
Know basic microbiology lab safety rules (a copy of this is available on Canvas) – eg. What
should be done in case of a bacterial culture spill.
If the bacterial culture got spill, cover any culture spill with paper towels, soak the towels
immediately with disinfectant, and allow them to stand for 15-20 minutes.
Lab manual P.3-P.5
Review Exercise 1-2: Glo Germ Hand Wash System
Microscope Ex 3-1
o Parts and uses of each part (Lab Manual P.125)
o Magnification of the oculars and objectives
Oculars: 10X
Objectives:4-5, 10, 40, 100
o How to calculate total magnification: Objective lens X Ocular Lens
o How to use oil immersion objective: Lab Manual P.129 #12
o Review the questions on the Microscope Lab Assignment that was given to you in class.
Cyanobacteria
o Be able to identify microscope slides of cyanobacteria (Nostoc, Oscillatoria, Gloeocapsa)
Nostoc
Oscillatoria
Gloeocapsa
Bacteria
o Recognize coccus, bacillus, spiral shapes of bacteria on microscope slides.
Cocci (Coccus) round shape Bacilli (Bacillus) Rod shape Spirilla (Spiral shaped)
Spirochetes (tightly coiled Vibrio (Comma shaped)
like a spring
o Be able to identify bacterial arrangements –
1. Strepto- Chain
2. Staphylo- Cluster
Fungi
o Recognize microscope slides of Rhizopus, Penicillium, Aspergillus, Saccharomyces
(yeast), Candida albicans
Rhizopus
Sexual Spores
Asexual Spores
Penicillium
Asexual Spores
Asperguillus
Saccharomyc
es (yeast)
Candida
albicans
Common cause of virginal
infection
o Know the type of spores (names of spores) formed by Rhizopus, Penicillium, Aspergillus,
and whether they are sexual or asexual spores.
o Know the infection caused by the fungus Candida albicans.
Virginal infection
Exercise 1-4: Aseptic Technique
o Thoroughly review the exercise on aseptic techniques and inoculation methods.
o Be able to answer the following questions -
Why is the inoculating loop flamed before and after use?
To kill microbes
Why is the mouth of the test-tube passed over the flame after opening and before
closing the tube cap?
To avoid the possible contamination
Why should Petri dishes be incubated in the upside-down or inverted position?
So the condensation drops won’t fall on the agar surface
In which hand should the tube cap be held while transferring cultures from one
medium to another?
In the loop hand
Exercise 1-5: Streak Plate Method of Isolation
o Review quadrant streak technique (Fig. 1-31) – procedure and use
o With regards to bacterial growth, what does the term pure colony or colony-forming unit
mean?
Pure colony (colony-forming unit) means a visible cluster of bacteria on a petri dish that
originated from a single bacterial cell, meaning all the cell within that colony are
genetically identical.
o Be able to identify what a good quadrant streak looks like.
o Know the reasons for the following mistakes when looking at a quadrant streak plate
If bacteria are found growing on the first streak area, and none in the 2nd, 3rd, and
4th streak areas.
Didn’t touch the first streak area at all or the first streak area get very diluted at
the end
If plenty of bacterial growth is seen in all the 4 regions of the quadrant streak.
Initial inoculum is too concentrated
Exercise 3-4: Smear Preparation and Simple Stain
o Review procedure for making a bacterial smear from liquid media and solid media (Fig.
3.39)
For the solid media, you need to use a loop of water to spread on the smear
o Explain the purpose of heat-fixing a smear.
To firmly attach a specimen, by killing the cell and adhering them to the slide. Thus
preventing them from washing off during the stain process.
o Know the stain that you used in the lab for the simple stain procedure.
Safranin/Crystal Violet/ Methylene Blue
Exercise 3-6: Gram Staining
o Review the steps & exact stain sequence of the Gram staining procedure (Fig 3.54).
1) Prepare for the bacteria smear( water/air dry/ heat fixed)
2) Apply Crystal Violet (primary stain) 1 minute
3) Wash with distill water
4) Iodine as mordant (fixed prime)
5) Wash with distill water
6) Ethyl alcohol for 10-20 seconds decolorizer
7) Wash with distill water
8) Counterstain with Safranin (pink) for 1 minute
9) Wash with distill water
10) Look at the smear under microscope from low-high-and then oil immersion
o Know the function of each substance used for gram staining.
Crystal violet- primary stain
Iodine- Mordant (fixed the prime stain)
Ethyl Alcohol- decolorizer
Safranin- counter stain
o Be able to recognize Gram-positive & Gram-negative bacteria under the microscope, and
identify their shape.
Gram-positive: Purple/Blue
Gram-negative: Pink
Tiny Bacilli Pink G-
Escherichia Coli
Round Cocci in purple G+
clusters
Staphylococcus Aureus
Bacillus Subtilis Rod-shaped Bacilli purple G+
Exercise 3-9: Endospore staining
o Know the name of the stain procedure used in the lab. Schaeffer-Fulton method
o Know the procedure and stains used for endospore staining (Fig 3.68).
1) Prepare the smear, air dry, heat fixed.
2) Apply malachite green stain on the smear, steam it for 5 minutes. Adding more
malachite green during the steaming if it dry out
3) Wash off the smear by Stillwater
4) Counterstain with Safranin stain for 1 minutes. Rinse with Stillwater
5) Gently blot dry with bibulous paper, do not rub.
6) Observe with oil immersion lense
o Know the stain that stains endospores and the stain that stains vegetative cells.
Endospore: malachite green
Vegetative cell: safranin
o Explain the purpose of steaming the slide during the staining process.
To force the primary stain (malachite green) into the tough outer coat of the endospore,
allowing it to penetrate and be visualized under a microscope
o Be able to identify vegetative cells and endospores on a microscopic slide.
Exercise 5-24: Motility determination
o Know to interpret the motility results by looking at a stab culture on semi-solid agar.
(Example of non-motile bacteria – Micrococcus luteus; Example of highly motile bacteria
– Proteus mirabilis)
Proteus mirabilis Staphylococcus Aureus
Motile(+) Nonmotile(-)
o What is the agar concentration in a typical nutrient agar medium and in semi-solid
medium? 1.5% vs 0.4%
Exercise 3-7: Acid-fast staining
o Know the name of the stain procedure. Ziehl-Neelson (ZN) Method
o Know the procedure and stains used for acid-fast staining (Fig. 3-58), & results of the
staining.
1) Apply Carbolfuchsin as primary stain (pink) (acid fast)
2) Steam (mordant) for 5 minutes
3) Wash with distill water
4) Wash with Acid Alcohol for 15-20 seconds to decolorize
5) Counterstain with Methylene Blue (non acid fast) for 1 minute
6) Wash off with distill water
o Be able to recognize microscope slide of acid-fast & non acid-fast bacteria.
o Know the stain that stains acid-fast bacteria and the stain that stains non acid-fast
bacteria.
o Why is Mycobacterium acid fast?
Because of peptidoglycan + waxy mycolic acid in the cell wall
Exercise 2-6: Fluid Thioglycollate Medium
o Know to distinguish an aerobic bacterium from a facultative anaerobe by looking at the
thioglycollate test results.
Pseudomonas aeruginosa Escherichia Coli
(Obligate Aerobe- growing only at the top, (Facultative Anaerobe- growing at O2 and
the O2 zone) none O2 zone)
o What is the purpose of adding thioglycollate to the medium?
Remove oxygen and create a anaerobic condition
o What is the indicator used in the medium?
Resazurin, this dye turns pink in the presence of oxygen
o Know how bacteria are classified based on the oxygen requirements.
Facultative Anaerobe- growing with or without oxygen
Obligate Aerobe- only growing with oxygen
(more classification in the lab manual p.81 but professor only wrote down these two)
