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Microbiology lab midterm

STUDY
PLAY
microscope
arm, base stage, substage light, condenser, iris diaphragm, coarse adjustment knob, fine adjustment knob, objectives, nosepiece, ocular lens
oil immersion
the use of a drop of oil, that has the same refractive index as glass to channel more light waves into the objective
total magnification
ocular magnification x objective magnification
cocci
round shaped bacteria
bacilli
rod shaped bacteria
spirochete
corkscrew shaped bacteria
spirillum
wavy shaped bacteria
bacterial capsule
negative stain- repelled by capsule.
types of flagella
endospores
gram positive / gram negative stains of bacteria
gram positive stains purple/ gram negative stains pink
staphylo- /strepto- / diplo-
staphylo- means grapelike clusters; strepto- means twisted chain; diplo- means double or two
acid fast stain
used for Mycobacterium tuberculosis
Penicillium
Rhizopus
Aspergillus
Saccharomyces
Balantidium coli
Plasmodium vivax
Trichomonas vaginalis
Giardia intestinalis (lamblia) cyst and trophozoite
steps in aseptic technique (forceps)
forceps: place the ends of the forceps into the beaker filled about 2 inches deep with ethanol. Hold the end of the forceps and pass the tips through the flame. The alcohol will burn off (it will flame). Make sure you do not tip the forceps up or down. Once the flame has gone out (not before or the entire beaker will catch fire), you will dip the forceps again in the alcohol and pass them through the flame. Generally three passes of the forceps though the flame are used to sterilize them.
steps in aseptic technique (test tubes)
The lid of the test tube is removed with the little finger of your dominant hand. Hold the innoculating loop in your dominant hand with your index finger and thumb, and the cap of the test tube with your little finger. Use your other hand to twist the bottom of the test tube until it's open. The test tube cap will be held in your little finger the entire time the test tube is open. As soon as the tube is open, pass the tip of the test tube through the flame. The loop is used to remove bacteria and the lip of the tube is then flamed again before the cap is put back on.
steps in gram staining
Prepare a smear of the bacteria onto the slide. Heat fix the slides. Then you are ready to gram stain. Place the slide with the heat fixed bacteria on the staining rack and cover the bacteria with crystal violet. Leave the crystal violet on for 1 minute. Rinse with distilled water being careful to not squirt it directly onto the bacteria. Cover the bacteria with the gram iodine mordant for 1 minute. Rinse again with distilled water. Rinse the bacteria with the gram decolorizer until the solvent flows colorless from the slide (about 5-10 seconds). Rinse immediately with distilled water. Overlay the gram safranin for 30 seconds. Rinse with distilled water and allow to dry.
general rules for aseptic technique
Always work close to the flame, do not talk while you have open containers of microorganisms, wear gloves, wipe table with bleach, label all beakers, tubes, etc.
descriptive terminology for agar culture
rhizoid: root like
umbonate: raised in center
punctiform: small individual colonies
spread: covering entire surface
arborescent: branching
beaded: tiny colonies
descriptive terminology of growth characteristics
elevation: flat, raised, or convex
surface: wrinkled, rough, dull, shiny
pigmentation: red, yellow, cream or white
opacity: transparent, translucent, opaque
size: measure diameter in mm
shape: circular, oval
edge: entire, lobate, erose, undulate, filamentous
descriptive terminology for broth
turbid: cloudy
flocculent: growth in clumps
sediment: growth at bottom
pellicle: growth around top rim
Steps of scientific method
streak plate
used to produce individual colonies of bacteria.
how to make a streak plate
begin with petri dish with TSA agar and draw three sections. Obtains a TSB of bacteria. Loosen the lid. With a sterile cotton swab, aseptically remove a sample of bacterial culture. Close the TSB tube and place back into rack. Working near the flame, hold the petri dish in your non dominant hand and glide the swab gently and rapidly in a zig zag pattern across the agar. Don't make a spread plate. Just zig zag five or six times across one third of the agar. Close the dish, flame the loop and cool. Streak the loop gently across the agar in the primary area to carry the organisms to the second section. Zig zag back and forth overlapping the primary area for the first three strokes. Flame and cool the loop. Now do the same for the second and third area. Flame the loop and invert the plate to store in the incubator.
spread plate
creating an entire yard of solid bacteria
how to make a spread plate
label the plates with the names of the bacteria you are using. Write your name and date on the bottom of plate. your goal is to make a solid yard of bacteria. Use a broth culture of each bacteria. Open the broth tube and flame the lip. Take out the cotton swab and dip the swab into the culture. Make sure you flame the lip of the tube before you close the tube. Use the swab to fill 3/4 of the plate with very spaced zig zag lines. The part immediately under your wrist will remain empty at first. Rotate the agar 1/4 turn and also turn the swab 1/4 and repeat. Repeat the procedure two times. Make sure not to gouge the agar. Discard the swab into a beaker of disinfectant. Place the petri dish upside down in the incubator.
steps of protein synthesis
protein synthesis begins with DNA. A phosphate and sugar backbone and the base pairs are linked to one another through hydrogen bonds. The backbone chains are arranged in antiparallel fashion to each other. The strands have a 5' end and a 3' end. The 5' end is the phosphate end (because phosphate is attached to the 5 sugar carbon) and 3' end is the sugar end (where an OH group attaches). The entire structure is wound into a helix. DNA replication is essential if a cell is to divide into identical daughter cells. This is called semiconservative replication because each new double helix contains one "old" strand as well as one "new" strand. The enzyme that adds the nucleotides, DNA polymerase, can only work in the 5'to3' direction.
Use of autoclave
121 degrees Celsius for 30 minutes at 15 lbs/in2. Used in hospitals to sterilize objects that can withstand pressure and moisture.
sporulation
process used by bacteria to produce endospores when they are exposed to adverse conditions. (no reproduction during endospore stage- however they are highly resistant, dormant structures) ex. Bacillus and Clostridium
germinate
process used by bacteria when conditions return favorable- stage after endospore
bacterial capsule
cell membrane, cell wall, glycocalyx (capsule/slime layer)
aseptic technique
technique used to avoid contamination.
mixed culture
several species of bacteria growing on a culture
pure culture
one species of bacteria growing on a culture
Gram stain
primary: crystal violet. Is a simple stain that will stain both gram +/- bacteria purple.
mordant: iodine. Added to crystal violet, it forms large crystals by binding with the crystal violet in the cytoplasm of the cells.
decolorizer: alcohol. Alcohol creates holes in the thin layer of peptidoglycan of gram - bacteria and completely dissolves the lps layer. Gram + are not affected because the alcohol makes the thick peptidoglycan less permeable to the mordant crystal violet crystals.
counterstain: safranin. Counterstain will stain the decolorized gram - bacteria pink.
zone of inhibition
the inhibition of bacterial growth around the disc of disinfectant. Measured in mm.
disinfectant
used for surfaces. Kills pathogenic bacteria.
antiseptic
used for skin. Kills pathogenic bacteria.
sterilization
kills all bacteria
transcription
making complimentary RNA strand from DNA strand- known as mRNA which takes copy of DNA to ribosome.
genetic code
the code on mRNA- which is codons (complimentary to DNA and anticodons- on tRNA). Each codon codes for a specific amino acid.
translation
process of making a protein from the codons in mRNA.
parts of a micropipette
plunger button/adjustment dial
tip ejector button
digital volume indicator
attachment point for disposable tip
sizes of micropipettes
0.5 ul- 10 ul
5 ul - 50 ul
20 ul- 200 ul
100 ul- 1000 ul
to increase the volume on the pipette
counterclockwise
to decrease the volume on the pipette
clockwise
dna fingerprint
unique sequences of nucleotide base pairs
restriction enzymes
enzymes that "cut" DNA between the G/A in the sequence GAATTC ( or another particular sequence)
agarose gel electrophoresis
technique for separating DNA fragments. Voltage of 200V for 60 minutes.
DNA has an overall ___________ charge
negative
nucleotide is made of
sugar, phosphate, and a nitrogenous base
loading dye
allows you to see bands of migration of DNA on the agarose gel- allows allows sinkage.
endonucleases
restriction enzymes produced by bacteria that can cut the DNA of other organisms into tiny pieces thus killing the organism.
transformation
process by which one cell takes up an extra naked piece of DNA that it didn't have previously
competent
ability to take up DNA.
CaCl2 (calcium chloride)
helps bacteria become competent
plasmids
small pieces of DNA located outside the circular chromosome
pGLO plasmid has
gene to make araC
GFP genes (replacing the arabinose genes)
bla gene (beta lactamase- makes bacteria resistant to ampicillin)
+pGLO/LB/AMP
grow/no glow
+pGLO/LB/AMP/ARA
grow/glow
-pGLO/LB/AMP
no grow/no glow
-pGLO/LB
grow/no glow
heat shock
42 degrees Celsius for 50 seconds (used along with CaCl2 to make bacteria competent)
arabinase
enzyme needed to break down arabinose
purpose of protein araC
needed to bind specific promoter that is needed to transcribe the genes for the production of the enzyme arabinase
colony
visible growth of bacteria that arises from a single progenitor.
two types of fungi
yeasts (uni); molds (multi)
hyphae
filaments or branches
spores
reproductive structures of molds
mycelium
made up of spores and hyphae (tangled mess)
yeasts
facultative anaerobes----> produce CO2 and H2O aerobically and produce ethanol and CO2 anaerobically.
molds
aerobic. Make penicillin. Easily inhaled and cause allergies, asthma, or respiratory diseases.
fruiting body
A specialized spore-producing structure, especially of a fungus. A mushroom is the fruiting body of a fungus.
schizogony
nucleus divides several times before the cell dividies creating a structure with two or four nuclei.
trophozoite
active feeding stage
cyst
hardy resting stage- has a protective coating or capsule that can resist harsh environmental conditions
protozoan reproduction
can occur through asexual budding, fission to sexual reproduction or conjugation
Semmelweis
importance of handwashing and the transmission of bacteria in a hospital setting
Staphylococcus epidermidis
usually non pathogenic except in immunocompromised patients. Bacteria are resident bacteria- they live in a mutualistic relationship with us
Staphylococcus pyogenes or aureus
transmitted from one human to another through skin contact. Most common contact with hands.
methods used to count bacteria
spectrophotometry, quantification of bacterial products, hemocytometry, and serial dilution
aliquot
specific volume of one tube (in dilution count)
proper pipetting technique
obtain micropipette, adjust volume by rotating dial at top. Insert attachment point of the pipette into a disposable tip. Depress plunger to the first stop. Hold pipette vertically. Slowly release plunger button. Water will rise in tip. Withdraw tip from water. To depress the water, depress plunger button to the second stop.- this ensures that all water is released. Release disposable tip into the tip jar by depressing tip ejector button.