Micro Lab Practical Flashcards
What are some factors that would have effected the microbial growth?
Time, temp, incubation, nutritional requirements
What is the main point of ubiquity?
understand that microbes are found virually everywhere
Proper set up of the microscope
-Use two hands to carry -Place gently on lab bench -Take off dust cover, put in cabinet -Unwrap cord completely -Make sure light is off and rheostat is turned down -Plug in cord -Turn on light
Proper storage microscope
-turn off light -Turn rheostat down -click low power lens in place over condenser -make sure there is no oil on lens or stage -lower stage all the way down -wrap cord around base but not condenser -tuck plug into coiled cord carefully put microscope into cabinet w/ dust cover
Total magnification
Ocular power x objective power
A focus issue, when moving from objectives the new image being relatively in focus
Parfocal
The virtual image appear upside down and backwards in the microscope
Inversion
Number of times the size of the image exceeds the original object
Magnification
Bending of light back into the field of view
Refraction
Given by the objection
Real image
Is seen through the oculars
Virtual image
Arm
Connects tube to base, used to carry the microscope
Base
Bottom support of the microscope
What microscope has two oculars
Binocular microscope
Body of microscope
Includes eye piece and objective lens but not the focus block
Course focus knob
Used to move objective lens towards or away from the specimen
Condenser lens
Increase elimination and resolution Focus or condense the light into a specimen
Thickness of object space within which objects focus by a lens will appear in good simultaneous focus
Depth of field or depth of focus
It varies the amount of light passing through the stage opening which will help to illuminate the specimen and increase contrast and resolution
Diaphragm
Knob used to focus on various parts of the specimen to fine tune the focus
Fine focus knob
Diameter of the circle of light that you see when looking through a microscope
Field of view
Moving the specimen closer or further away from the objective lens to render a sharper image
Focus
Special oil used in microscopy with only the 100x objective lens. Acts as a bridge between the glass slide and the lens which concentrates the light path and increases the resolution of the image
Immersion of oil
The adjustment made for the distance of the viewers eyes which allows the eye piece lenses to spread apart of get closer together. It allows the viewer to see the specimen with both eyes
Inter pupillary adjustment
Consists of a slide holder and two knobs. One know moved the slide towards or away, and the other knob moves the slide left and right
Mechanical Stage
Also called a micron is the metric linear measurement used in microscopy. 1/1 millionth of a meter
Micrometer
Part of the microscope that holds objective lenses
Revolving nose piece
Lenses that are closest to the object and gives the 3-D effect
Objective lens
When changing from one objective lens to another the image of the object should stay centered
Par centered
Ability of a lens system to show fine details of the object being observed
Resolution
How to prepare a bacterial smear:
-Mix liquid culture -Place two drops on center of slide using loops -spread to app. Quarter size -air dry completely -heat fix
Why do we air dry completely?
So that the cells can shrink
What are three reasons to heat fix:
-kills any alive bacteria -facilitates stain penetration -fixes the cells cell wall to the slide so they don’t wash off when staining
How many stains are used in simple staining?
1 - crystal violet
What is a basic stain and why is it used?
Crystal violet is the basic stain and is used because it has a positive charge which is attracted to a negative cell wall
What is the charge in a bacterial cell?
Negative charge
New cell shapes and arrangements
Palisades- bacilli | | Sarcina- a cocci forms a 3D cube
The 4 steps of gram stains?
- Crystal violet 2. Iodine 3. Alcohol 4. Saffrinin
What’s the function of crystal violet?
Primary stain
What is the function of iodine?
It’s a mordant
What is the function of alcohol
De-colorizer
What is the function of saffrinin?
The counter stain
The colors for a gram positive in a cell in each step:
1.crystal violet- purple 2. Iodine- purple 3. Alcohol- purple 4. Saffrinin- purple
The colors for a gram negative in a cell in each step:
- Crystal violet- purple 2. Iodine- purple 3. Alcohol- colorless 4. Saffrinin- pink
What could go wrong in a gram stain?
-Too much decolorization of gram positive may cause them to lose its color and appear gram negative -insufficient decolorization of gram negative may allow them to retain their purple color and appear positive
Why is gram staining more widely used than simple staining?
Since most bacteria are gram +/- so we do gram stain to differentiate between them
What is the structure that is being differentiated in gram staining?
Cell wall
How do gram-positive and gram-negative organisms differ structurally?
Gram-negative have less peptidoglycan. Therefore, fail to trap the iodine Crystal violet complex. Also has an outer membrane and two periplasmic spaces.
Why did we use the modified Anthony’s procedure in capsule staining?
Acid stains do not look good
Purpose of the milk broth in capsule staining?
To give a stand-able background
Function of a capsule for a bacterial cell?
- Protection from phagocytosis
- serves as a buffer between the cell and it’s external environment
- protects bacteria from dehydration and traps nutrients from surrounding environments.
What are the dangers of heat fixing in capsule staining?
We can get burned but also dehydrate the cell and creates a false capsule where the stain would not bind
Function of endospores
Ensure survival of bacteria
Different endospores presentation
-terminal, non-swollen, circular -sub terminal, non-swollen, circular -central, non-swollen, circular -terminal, swollen, circular -sub terminal, swollen, circular -central, swollen, circular -free, circular -free, oval
How and why is the bacticinerator used, in transfer techniques?
To sterilize the loop during microbial transfers in order to prevent contamination.
Movement of bacteria from one media to another
Subculturing
Contains only 1 species
Pure culture
Contains more than one microbial species
Mixed culture
Movement of microbes without contamination
Aseptic technique
Introduction of unwanted microbes
Contamination
Separate masses of bacteria
Colony
How many cells does a colony originate from?
1
Evidence by turbidity
Broth growth
Types of broth growth
Ring Pellicle Sediment Uniform fine turbidity Flocculent
Evidence by visual mass of growths as well as pigmentation & optical properties
Slant growth
Types of slant growths
Filiform Spreading edge not eagle Friable
Non-motile and lacks flagella
Filliform
Motile with flagella
Spreading edge not eagle
Dry and crusty!
Friable
The most information is provided by plate growth, what elements are examined?
examine form, margin, elevation, optical/pigment properties
________ tells you the overall shape
Form
Form types:
Circular Irregular Punctiform Rhizoids Filamentous
Rise off the agar surface
Elevation
Elevation types:
Flat raised convex pulvinate umbonate crateriform
Describes the edge if colonies
Margin
Margin types:
Entire Lobate Undulate Curled Rhizoids Filamentous
Describes color
Pigmentation
Pigment types
Pigmented Non-pigmented
Optical properties describe
reflected and transmitted light
Reflected light types:
Shiny- rough, smooth Dull- rough, smooth
Transmitted light types:
Opaque- no light can pass through, milk Translucent- some light passes, apple juice Transparent- all light passes, water
Organisms have ranges in which they can grow: minimum, maximum, and optimum
Cardinal temperature
Cardinal temperature determines ________
Thermal classifications
Types of thermal classifications:
Psychrophile Mesophile Thermophile
Psychrophile thrive in
Mesophile thrive
Between 20-45 deg Celsius
Thermophiles thrive:
Above 45 deg Celsius
______ may be used to kill microorganisms on inanimate objects such as instruments and thermometers.
Disinfectants
Used to kill organisms on living organisms such as skin
Antiseptics
Form
Circular
Elevation
Convex
Elevation
Pulvinate
Elevation
Umbonate
Elevation
Crateriform
EleelElevationEleEElElevation
Flat
Elevation
Raised
Form and Margin
Filimentous
Form
Irregular
Form
Punctiform
Form and Margin
Rhizoid
Margin
Entire
Margin
Lobate
Margin
Undulate
Margin
Curled
Margin
Filimentous
Pigmentation
Pigmented
Pigmentation
Non-Pigmented
Reflected Light
Dull, Rough
Reflected Light
Dull, smooth
Reflected Light
Shiny, rough
Reflected Light
Shiny, smooth
Transmitted Light
Opaque
Transmitted Light
Translucent
Transmitted Light
Transparent
What does heat do?
denature proteins
What do cold temperatures accomplish?
slow or inhibited growth
Effects of moist heat
decrease temperature & decrease exposure
Effects of dry heat
increase temperature & increase exposure
What is an antibiotic?
naturally produced by bacteria and some fungi
What is an antimicrobic?
partially or fully synthesized in the lab; improvements made to antibiotics to help us
What were the limitations of the temperature experiment?
too few temperatures
ideally need 1 degree incraments
no identifiable cardinal range- min max optimum
Where can susceptible organisms not grow?
in the zone of inhibition
Where can resistant organisms grow?
there is no zone of inhibition; so they can grow everywhere
_____ _____ works on a small range of microbes
narrow spectrum
_____ _____ works on a wide range of microbes
broad spectrum
Why is quadrant streak plating used?
to separate out cells in a mixed culture which allows for visually distiguish various bacteria in order to relocate for a pure culture
What is the procedure for simple staining?
flood slide with crystal violet; allow to sit for 60 seconds; rinse with H2O; blot dry with bibitous paper
What is the proper technique of handwashing?
warm water; plenty of soap; hands angled downward; say ABC two times; rinse; turn water off with paper towel
Resons for non-compliance in hand-washing?
busy, soap is irritating to skin, gloves are hard to put on
How do you make a bacterial smear?
two drops of water on slide with loop; spread bacteria from slant/broth/plate in H2O; air dry; heat fix
What dye is used in endospore staining?
malachite green & safranin
What type of stain is the endospore stain?
structural stain
LIst several factors that must be standardized to enhance the usefulness of the antibiotic susceptibility test by the Kirby-Bauer method
depth of agar, pH, number of microbes
Everything standardized except the drug effect to be examined
Why is it more advisable to use a swab instead of a loop for preparing a lawn of bacteria on an agar plate?
the loop can cut the agar and is less effective while a swab makes it easier to spread and has less damage potential
How might one explain the appearance of colonies of bacteria within the zone of inhibition?
resistant mutants have grown
What does the Kirby-Bauer method test for?
how to test for anti-microbic drugs
What type of agar was used in the Kirby-Bauer method? What makes it unique?
Mueller Hinton;
it is an H2O based agar that allows for diffusion through membranes and growth from an extremely broad range of organisms
What could happen if a bacterial smear is too thin?
the cells might be hard to find
What could happen if the bacterial smear is too thick?
it could be hard to see individual cells
Why is heat necessary for the successful performance of the spore stain technique?
bacterial endospores contain numerous protective layers which cannot be penetrated easily, so heat is used to drive the dye into the cells
HOw is the effect of antibiotics on bacteria determined?
Standardized Disk Susceptibility Test
a.k.a Kirby-Bauer Method
What are the two functons of CuSo4 in the ANthony Stain Procedure?
staining and rinsing the dye away
What was the purpose of the control in the temperature experiment?
tested comparison which shows the effect of no growth o bacteria after heat exposure
