Microbiology Lab 1232 Exam 1 Study Guide

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Exercise 1 and 2

Question Answer
aseptic technique (def'n)collection of procedures and rules used to minimize the possibility of microorganismic contamination
Steps to Aseptic Technique1. Arrange the tube with the live culture in V formation..2. use other hand to flame the loop..3. hold loop in air 20 sec..4. use inoculation hand small finger to uncap tube..5. flame mouth of tube..6. get culture..7. reflame tube
transient organismsreadily killed by common antiseptics
What are transient organisms also called?contaminating
How are transient organisms removed?removed by common antiseptics..are generally completely removed by 7-8 minutes of washing with soap and water
Resident organismsmore difficult to remove by antiseptics or washing
Ways to prevent contamination1 - invert Petri dish to prevent condensation from contaminating culture..2 - Don't touch the inoculating loop (ILP) to any surface while transferring specimens..

Exercise 3 The Microscope

Question Answer
Brightfield Microscopemicroscope which allows light rays to pass directly from from the light source, through various other components of the microscope, to the observer's eye
working distancephysical distance between the lens and the slide or cover slip when the object is in sharp focus on that objective lens
total magnificationmultiply the magnification of the objective lens that is in position by the magnification given by the ocular lens
magnification1 - one of the main functions of a microscope..2 - defined as increasing the apparent size of the image
resolution1 - one of the main functions of a microscope..2 - defined as improving the clarity of an image
parfocal"equal" + "pertaining to focus"..means that when the image is in sharp focus on any objective lens, you can switch to any other objective lens without moving the stage at all and the image should still be in good focus with only fine adjustment necessary
parcenterwhen the image is in the center of the field of view on any objective lens, it will remain in the center when you switch to any other objective lens
Ocular Lensbrings the image from the objective lenses to the observer's eye (10X magn)
objective lenslocated above the mechanical stage, magnify the image of the object being viewed
10xlow-power, used for initially locating the specimen on the slide (yellow band)
40xhigh-dry (blue band)
100xoil-immersion lens (white band)
Basethe part on which the microscope rests during operation
Armthe part which holds together the base, the stage, and the various optical components of the microscope
Illumination sourceprovides the light for viewing specimens on the microscope
coarse adjustment knobregulates the position or the stage...up and down...only used with low-power lenses
fine adjustment knobused for precise focusing...after coarse adjustment...high power lens use fine adjustment only
iris diaphragmregulate the amount of light that can enter the condenser
stagepart onto which the specimen is placed

Exercise 3 part 2

Question Answer
What does the resolving power of a microscope depend on? 1 - design of the condenser 2- quality of the lenses 3 - wavelength of the light used 4 - use of immersion oil with the immersion lens
What is the resolving power of the best commercially available microscopes?0.2 micrometer
What is the purpose of the immersion oil?The immersion oil has the same refractive index as the glass, so when the drop is placed between the slide and the oil immersion lens, there is no refraction to cause the image to blur
For best resolution, the condenser should be ___as high as possible...the iris diaphragm should be completely open..and immersion oil should be used
Precautionscarry with two hands...make sure light source "off" before plugin..wipe oil immersion with 70% ethyl alcohol..never use coarse adjustment with 40x or 100x
What are the steps to preparing a wet mount?see
What are the steps to preparing a hanging drop slide?see

Exercise 4 Subculturing

Question Answer
colonya group of cells which arises from a single cell
subculturingstarting new colonies from the isolated colonies
streak plate methoda common method for isolating bacteria fro a mixed culture. The objective is to "thin out" bacterial growth in successive sectors of a Petri dish until individual colonies are obtained.

Exercise 4 Steps to Streaking for isolation

Item Next
1 - aseptic technique applies in each step2 - make zigzag vertical streak along one edge
2 - make zigzag vertical streak along one edge3 - flame loop
3 - flame loop4 - beginning at edge of vertical streak create a perpendicular streak along the 2nd edge of the Petri dish
4 - beginning at edge of vertical streak create a perpendicular streak along the 2nd edge of the Petri dish5 - flame loop
5 - flame loop6 - beginning at edge of vertical streak create a perpendicular streak along the 3rd edge of the Petri dish
6 - beginning at edge of vertical streak create a perpendicular streak along the 3rd edge of the Petri dish7 - flame loop
7 - flame loop8 - make a fourth streak at the end of the third toward the center of the dish
8 - make a fourth streak at the end of the third toward the center of the dishlast

Exercise 5 Preparing Smears and Simple Staining

Question Answer
heat fixpassing the slide rapidly through the flame of the Bunsen burner...heat fixing will partially affix the bacteria to the slide, making them much more difficult to remove during the subsequent staining and washing
direct staina stain of the bacteria itself...basic stains are used in direct staining
negative staina stain of the background of the bacteria
agarsubstance used in culturing microorganisms..melts in water at 85 degrees C..remains liquid until cooled to about 40 degrees C..Once solid, it can be used at temperatures from 10deg C to 40 deg C..
why is agar a great medium for growing bacteriait resists degradation by the bacteria
where does agar come from?agar is a polysaccharide which was initially extracted from the marine red algae
agar will form a solid medium at about 0.8% concentration
an agar concentration of ___ will be completely solid1.5 to 1.8%
because of its great water holding capacity (of agar)scientists can dissolve many nutrients into the agar and still have a solid medium
What is semi-solid agar good for?pour plate counting of bacteria...motility assays..etc.
agar plates are (good / not good) for the long term storage of bacterianot good..dry out quickly and easy to contaminate due to large surface
What two media are used for long term storage of bacteria?agar slants and agar deeps
agar slanta tube in which the agar has been solidified at an angle
agar slants are good for storing bacteria for ___ ___3 months (or longer)
agar deeptues in which agar is solidified in a horizontal line
agar deeps are good for storing bacteria for ___up to 6 months
agar deeps are also used when the organism cannot tolerate ___.oxygen

Exercise 5 part 2

Question Answer
basic stains have a ___ chargepositive
What charge is carried by acidic stainsnegative
Most components of a bacterial cell are ___ chargednegatively charged
What is another name for dyes?chromophores
Why don't acidic stains stain the bacterium?the acidic stain is negative and the bacterium cell wall is negative, so the charges repulse each other
Which stain (basic or acidic) would you utilize heat fixation?basic (direct stains)
Categories of stains - methylene bluebasic stain (direct stain)
Categories of stains - crystal violetbasic stain (direct stain)
Categories of stains - malachite greenbasic stain (direct stain)
Categories of stains - safraninbasic stain (direct stain)
Categories of stains - carbolfuchsinbasic stain (direct stain)
Categories of stains - Congo redacidic stain (negative stain)
Categories of stains - Nigrosinacidic stain (negative stain)
what is feathering?a technique of spreading a stain (mixed with specimen) in which you should have a smear which is faily thick at the starting point and fether-thin at the other end of the slide (see
define morphologyshape of microorganism
Under the microscope, what is the morphology of the E. coli?short rods
What does E. coli stand for?Escherichia coli
Under the microscope, what is the morphology of Micrococcus Luteus?a coccus
What is a coccus?round bacterium
Heat fixing is used in (positive / negative) staining?positive
Is heat fixing used in negative staining?No
Why is heat fixing not advisable in negative staining?the bacteria are fixed to the slide, as you smear, you would not have the bacteria within the stain
What are the steps to create an air dried heat fixed slide?see
What are the steps to create a negative stain?see

Exercise 6 The Gram Stain

Question Answer
differential staina differential staining procedure is one wher more than one stain (and sometimes more than one type of stain) is used to differentiate bacteria into groups
What type of stain is the Gram stain?differential stain
virtually all bacteria fall into what two groups distinguished by the gram stain?gram positive and gram negative
What creates the difference between gram positive and gram negative bacteria?the structure of the bacterial cell walls
The cell walls of gram positive bacteria contain significantly less ___, and are thus less sensitive to solvents
Due to the lower lipid amount within the cell wall, ___ ___ bacteria tend to retain the crystal violet complex even after ___ with 95% ethanol.gram positive...decolorization
Gram negative bacteria contain an outer membrane which is made up of ___.lipopolysaccharide (LPS)
LPS on gram negative bacteria (can / cannot ) be easily dissolved by ___.alcohol
Why do gram negative bacteria lose the crystal violet after decolorization by 95% ethanol?the outer membrane is dissolved easily by the 95% ethanol, due to it being comprised of LPS (lipopolysaccharide)
After decolorization, what was used in our lab to color the cell remaining?safranin (a direct or basic dye) in color
What are the steps to gram staining?see
What is the purpose of Gram's iodine?it helps affix crystal violet to the cell walls of Gram-positive organisms, making it much more difficult to remove
What does the Gram's iodine create?Gram's iodine will combine with the crystal violet dye in teh bacterial cell walls to form the crystal violet-iodine (CV-I) complex
If decolorization is excessiveeven Gram-positive organisms will lose the crystal violet and appear Gram-negative, when the stain is completed
If decolorization is too littleGram negative organisms may retain some crystal violet and appear Gram positive (because the outer lipid rich membrane is not dissolved
What are the steps to decolorization?see
At the end of the Gram stain, the Gram positive appeared ___ while the gram negative appeared
Would Congo red, another pink-red stain, be a suitable replacement for safranin in the Gram stain? Why or why not?No, because Congo red is a acidic (negative) stain, and the Gram stain uses two basic stains (positive stains). The positive and negative in Gram staining refer to the relative sensitivity to organic solvents, not charge.
Describe the color and morphology of the E. coli and M. Luteus after the Gram stainE. coli (gram +) are purple rods and the M. Luteus (gram -) are pink cocci

Exercise 7 Capsule Stain

Question Answer
secretions sometimes form a viscous less defined _________ layerslime
if the secretions form a well defined structurecapsule
What is the general term that includes both of the structuresglycocalyx
What are capsules composed of?a wide variety of materials including polysaccharides, glycoproteins, and polypeptides
What determines the exact composition of a capsule?genetic makeup of the bacterium and nutrients
What are the roles of capsules?elimination of metabolic wastes...prevention of dehydration (drying)...less vulnerability to phagocytosis
Give two capsule forming bacteriaStreptococcus pneumoniae and Bacillus anthracis
Do Streptococcus pneumoniae and Bacillus anthracis have the same infection ability if their capsule forming capability is hindered?no
Are capsule forming bacteria readily stained?no
While staining the K. pneumoniae in the Capsule staining is heat fixing applied?no
Take care not to ___ the bacteria off the slide, since they are not ___ ___.wash...heat fixed
What is the capsule forming bacterium used in this lab on capsule stains?Klebsiella pneumoniae

Exercise 7 Capsule staining steps

Item Next
Step 1 - prepare a negative stain of Klebsiella pneumoniae by mixing a loopful of the bacteria into a drop of Congo redStep 2 - spread the mixture over the slide using another slide, as you did in exercise 5
Step 2 - spread the mixture over the slide using another slide, as you did in exercise 5Step 3 - Air dry without washing or heat fixing
Step 3 - Air dry without washing or heat fixingStep 4 - Cover the Dry smear with Carbolfuchsin
Step 4 - Cover the Dry smear with CarbolfuchsinStep 5 - Let stand for 3 minutes
Step 5 - Let stand for 3 minutesStep 6 - Gently rinse the slide with physiological saline solution
Step 6 - Gently rinse the slide with physiological saline solutionStep 7 - Observe
Step 7 - Observelast