MICRO LAB PRACTICAL #1

1

occulars/eye pieces

2

body tube

3

nose piece

4

condensor lens

5

objective lenses

6

coarse knob

7

fine knob

8

stage

9

illuminator

10

iris diaphragm

function of occulars/eye piece

what you view through

function of body tube

houses the lens system that magnifies the specimens

functions of nose piece

revolves and contains the objectives

function of condensor lens

two lens system gets light from the illuminator and lets through iris diaphragm

function of objective lens

magnifies the specimen

function of coarse knob

moves stage up and down

function of fine knob

helps make fine tuned adjustments to see plate clearly

function of stage

holds the slide with a hole in the center to let light from the illuminator in

the stage often contains ___ ___ to hold the slide in place

stage clips

function of illuminator

the light source at the base microscope

function of iris diaphragm (below stage)

controls amount of light let in by the illuminator

what is the total magnification for the following objective lens: 4X

40x

what is the total magnification for the following objective lens: 10X

100x

what is the total magnification for the following objective lens: 40X

400x

what is the total magnification for the following objective lens: 100X

1000X

Which focus knob(s) should you use with the 4x objective?

coarse and fine

Which focus knob(s) should you use with the 10x objective?

fine

Which focus knob(s) should you use with the 40x objective?

fine

Which focus knob(s) should you use with the 100x objective?

fine (and immersion oil)

If you are unable to focus at the 40x, what should you do? Should you just skip ahead to the 100x?

no, go back to previous objective and focus

What do you need to add when transitioning from the 40x to 100x objective lens?

immersion oil

What steps can you take to troubleshoot a blurry image?

Clean lens, adjust diaphram, go back to previous objective

definition of parfocal nature of the microscope

when you change the objective lens it will stay almost in focus

Describe how to use the par-focal nature of your microscope when observing a sample?

switching to next objective lens you will only have to use the fine knob

definition of parcentral feature of microscope

specimen stays centered when switching magnifications

What is the purpose of adding oil when using the 100x objective lens?

Improves resolution of the image and reduces light refraction

How do you properly carry a microscope?

both hands one on base one on handle

What kind of paper do you use to clean oil off a microscope lens?

lens paper

When looking at your wet mount under the microscope what did you notice about the difference in size between the bacterial cells and the cheek cells? Which one was bigger? Blood cells vs bacterial cells?

cheek cells, blood cells, bacteria

identify the shape of each cell

cocci

identify the shape of each cell

bacillus

Add multiple choice options

diplococci

coccus

diplococci

clusters

pairs (diplobacillus)

rods (bacilli)

chains (of cocci)

tetrads (of cocci)

What tool should be used to inoculate a plate? What about an agar deep?

loop, needle

You are transferring a sample from the stock tube to 3 different plates. Should you incinerate your loop between each inoculation?

yes

Describe the steps you would take to ensure aseptic conditions when transferring microorganisms from one culture to another.

Incinerate loop, close lid, hold at 45 degree

What are ways contamination could occur when culturing an agar plate?

Leaving lid open, not incinerating loop

Which micropipette would you use to transfer 12 μL?

P20

Which micropipette would you use to transfer 198 μL?

P200

Which micropipette would you use to transfer 300 μL?

P1000

What four things are needed when labeling a sample?

name, date, section, culture name

How should your plates be stored? Should the agar be on the bottom or top?

upside down agar on top

What part of the microscope would need to be adjusted to increase or decrease the contrast during a wet mount? Where on the microscope is this part located?

iris diaphragm, under stage

less light from iris diagraph = ___ (more/less) contract

more

more light from iris diagraph = ___ (more/less) contract

less

Why would a coverslip be used to observe bacteria?

Stops bacteria from moving, prevents lens from contamination

Order the following organisms from largest to smallest: E. coli, S. cerevisiae, cheek cell

cheek cell, S. cerevisiae, E. coli

What are the advantages of using a wet mount?

see living organisms, movement, simple to prepare

What are the disadvantages of using a wet mount?

hard to see details, can dry out, bacteria can move

What is a pure culture?

a culture with only one type of bacteria

What would the result be if you were culturing an agar plate and forgot to let the inoculating loop cool between sections 2 and 3?

kill the bacteria, no growth on sections 2 and3

Based on what characteristics can one ensure they have obtained a pure culture on their plate?

colonies have same, shape, color, texture, form

What is this method called?

T streak method

What could happen if you overheat the slide during the heat-fixing process?

it can kill the bacteria

What could happen if you underheat the slide during the heat-fixing process?

cells may wash off the slide

What happens if you heat-fix a slide before drying it?

can kill the bacteria

Why is it important to heat-fix the slide before applying stains?

so the bacteria stays on the slide during washing

How can an improperly prepared smear affect your gram stain results?

can give false positives or negatives

Process of making a smear from a solid culture

add drop of water and mix bacteria in

process of making a smear from a liquid culture

place on slide

What is the term for the liquid used to promote primary stain retention in a gram stain? What solution has this function in a gram stain?

iodine helps the primary stain stick better

name for primary stain

crystal violet

function of primary stain

stains all cells purple

name for mordant

iodine

function of mordant

fixes dye into cell wall

name for decolorization

95% ethanol

function of decolorizer

removes dye from gram negatives

name for counterstain

safarin

function of counterstain

stains gram negatives pink

Interpret the following gram stain reaction and describe the morphology

gram positive cocci in clusters

Interpret the following gram stain reaction and describe the morphology

gram negative bacilli (rods)

If your known gram negative rods appeared purple under the microscope. What mistake in the staining process would cause this?

crystal violet stain or ethanol

name two errors that can occur during the staining procedure and what they result in when looking at the slide through the microscope

too much or little colorizer and can cause incorrect color

What happens if you let the ethanol sit on the slide too long? Not long enough?

can strip purple dye, cause gram negatives to be purple

what happens if your sample is too thick on the smear

cells too clustered to identify

What equation was used to determine CFU/mL in the Quantitation lab?

number of colonies/total dilution

Using the same steps as in the Quantitation lab, if your plate had a total dilution of 10-7 had 57 colonies, what is the CFU/mL in the original sample?

5.7x10^8 CFU/mL

Calculate the CFU/mL of the original sample if you followed the same steps as in the quantitation lab for the following plate: Number of colonies = 268 and Plate = total dilution 10-5

2.68x10^7 CFU/mL

?

10^-6

?

10^-8

What is the difference between direct and indirect quantitation?

counting, measuring turbidity

How would results be affected by an uneven distribution of bacteria over plates during the direct method of quantification of microorganisms?

inaccurate doesn’t represent the true concentration

Although we did not plate the 10-1 tube in our Quantitation lab, based on your results, what would you expect your 10-2 plate to look like if you had?

TMC