LAB 1
Answer the following questions
1. What three elements are used in an autoclave to sterilize equipment?
heat, pressure, and steam
2. What is the
... [Show More] minimum temperature an autoclave must be set at to achieve sterile condition?
125°C
3. If you are working in a lab in which an autoclave is not available, and you are pressed for time,
which would you chose to best sterilize your equipment? Hot steam or hot air? Explain why you
chose your answer.
Hot steam is the best choice as you can achieve a sterile environment in a matter of minutes
whereas hot air will take several hours to achieve the same effect.
4. What type of incubator is pictured below?
Fixed incubator
Answer KeyAnswer the following questions.
1. At what temperature is the fixed incubator set to, as presented in the lab video?
37°C
2. At what temperature should you refrigerate bacterial samples? Explain why this is beneficial.
4°C. This temperature slows bacterial growth and prolongs the life of the sample.
3. What are the FOUR types of gloves presented in the lab video?
Latex, Nitrile, Thermal cold, Thermal heat
4. What THREE rules were discussed in regards to lab safety that would protect you and others from
contamination?
1. Never eat or drink in the lab
2. Always wear appropriate PPE
3. Never leave the lab wearing PPE
5. What are the main sections that should be found in a lab notebook? Name at least 4.
Objective, Procedure, Notes, Results and Deviations
Answer the following questions.
1. You are a lab instructor and Sally Miller has turned in her lab notebook for you to grade the lab
experiment #1 on lab safety. Based upon what was covered in the lab video, how should Sally have
entitled her experiment?
SM01 Lab Safety
2. You arrive to your first day of work at a new lab. You are taking over for someone who took a new
job at another lab. Your boss informs you that because of time restraints, this person did not exactly
follow the experimental protocol. In order to proceed, you must know what he did differently. (1)
According to the lab lecture, under what section of his notebook would you look to find theexperimental steps? (2) As changes to the experimental steps were made, what are these
differences called and how should it appear in the lab notebook?
(1) Procedure—this is where the steps for the experimental protocol are recorded.
(2) Deviations. All deviations should be written in red to immediately bring attention to the
changes in the protocol.
LAB 2
1. Identify the part of the microscope indicated by the arrow.
Oculars (or eyepiece)
2. Identify the part of the microscope indicated by the arrow.
Objectives (or objective lens)3. Identify the part of the microscope indicated by the arrow.
Course focus
1. You are about to study a bacterial sample under a light microscope. You look into the oculars and
see two circles. What adjustments need to be made?
Compress or expand the oculars until a single circle can be seen while using both eyes
simultaneously.
2. What 2 parts of the microscope contributes to the total magnification to your sample?
Objective and oculars (eyepiece)3. As you looking through the microscope you wish to dim (or limit) the amount of light entering into
the eyepiece—what component of the microscope other than the light source itself can be adjusted
to make these modifications?
Diaphragm
1. What objective power is best suited if you are uncertain what the sample is and where to begin?
4x (or lowest power objective)
2. You are viewing a sample of bacteria that is 3 mm in diameter through a 40x objective lens. The
eyepiece has a magnification power of 10x. What size will the sample appear through the eyepiece?
1200 mm in diameter (or 400x’s larger)
3. Based on the microscope shown in the lab lecture, which objectives would NOT require placing oil
on the slide?
4x, 10x and 40x
LAB 3
1. List the 4 main steps used to prepare a DRY mount and indicate which step is optional.
1 – Clean slide
2 – Circle area on slide for specimen placement (OPTIONAL)
3 – Apply organism to slide
4 – Air dry at room temperature
2. Why is it important to first clean your slide before applying your sample?
You must first remove any unwanted contaminants from the slide otherwise it may be difficult
to distinguish between the pathogen of interest and a contaminant.
3. When performing a wet mount technique, what is the advantage of using a wax or hydrophobic
pen?Creating a hydrophobic barrier (the circle) helps to keep the water within the circle so it does
not spill off of the slide.
4. What dye do Gram-positive bacteria primarily retain?
Crystal violet
1. Why are Gram-positive bacteria able to retain the crystal violet dye?
They contain a thick peptidoglycan layer in their cell wall that readily retains the dye.
2. Identify the Gram status (positive or negative) and shape of the bacteria pictured below.
Gram-Negative; Bacillus (rod)
3. An acid-fast stain is most commonly used to identify what type of bacterium? What is the name of
the primary dye used in this technique?
Mycobacterium; carbolfuchsin dye
4. Why do bacteria repel the dye nigrosin?
Nigrosin is a negatively charged dye. The membranes of most cells are also negatively
charged. The membrane will repel the dye not allowing it to be absorbed.
1. What is one disadvantage of heat fixing a sample?The heat fixing procedure kills the specimen. This also prevents any observations on motility
and enzymatic properties.
2. What is the proper way to dispose of all materials used during the lab?
All materials must be place in a biohazardous waste bag and placed into an autoclave for
sterilization.
3. What are the Gram status, shape and identification of organism #2 from the Gram stain
procedure?
Gram-negative; Bacillus (rod); E. Coli
4. What are the Gram status, shape and identification of organism #5 from the Gram stain
procedure?
Gram-positive; Cocci (spherical) chain; Streptococcus
LAB 4
1. What type of media is best used to eliminate certain bacteria from within a mixed culture?
Selective media
2. According the lab module, what type of agar plate is the most commonly used nutrient agar? What
color is it?
LB agar; Light (or pale) yellow
3. What was the name of the selective agar plate (shown below) that is similar to a blood agar plate?
MacConkey agar1. What type of bacteria does MacConkey agar select for?
Gram-negative bacteria
2. Sorbitol MacConkey (SMAC) agar is:
A. Selective media
B. Differential media
C. Selective and differential media
D. None of the above
C
3.What pathogen is best identified using a SMAC agar?
E coli O157:H7. SMAC agar specifically differentiates between non-pathogenic E coli and the
pathogenic E coli strain O157:H7.
4. What is the purpose of doing a 4-phase dilution streak?
The purpose of a 4-phase dilution streak is to isolate individual bacterial colonies.
5. If you were required to grow 4 types of bacteria on a single agar plate without cross contaminating
the samples, what method would you use?
Quadrant growth method
1. Based on the 4-phase dilution streaking experiment, in what phase were bacterial isolates
(colonies) observed?Individual colonies were observed within Phase 3.
2. Identify the plating method (below) as demonstrated in the lab.
Quadrant growth
3. Identify the organism growing on the TOP half of the agar and describe the observed hemolytic
properties.
Staph Aureus; Beta hemolysis is observed based on the zones of clearing within the red agar.
4. Would you expect to see a color change when pseudomonas is streaked onto an EMB agar plate?
Explain your answer.
No. There would not be a color change because pseudomonas does not ferment lactose.Lab 5
1. The Kirby-Bauer method for examining antibiotic sensitivity is also known as what?
The Standardized Disc Susceptibility Test
2. True or False. The antibiotic discs are placed onto the LB agar plate before spreading the
bacterial culture on the plate.
False. The antibiotic discs are place onto the plate AFTER the culture has been spread.
3. When performing the Kirby-Bauer method the areas of clearing surrounding an antibiotic disc after
an overnight incubation are known as what?
Zones of Inhibition.
1. Why was an LB agar plate used to test the Staph culture as opposed to a selective/differential
agar that only grows Staph?
LB agar is used as it simply provides the nutritional requirements to encourage bacterial
growth. Since the results of the Kirby Bauer method is directly based on bacterial growth
patterns, no other selective or differential additives should be present that may hinder or
inhibit the samples growth.
2. What unit of measurement is used when determining the size of the zones of inhibition?
A. Centimeters
B. Micrometer
C. Millimeters
D. Meters
C
3. True or False. In order to maintain proper spacing the antibiotic discs should be place around the
edge of the plate.
False. The disc should be placed approximately a fingers width from the edge so that a
uniform zone of inhibition can be seen around the entire disc.1. Given the following image, determine whether the bacterial sample is resistant or susceptible to
the following antibiotics [Show Less]