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Micro Lab

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Found microbes under the microscope in the 1670's
Antonie van Leeuwenhoek
total magnification
4x * 10x
10x * 10x
40x * 10x
40x
100x
400x
When you first bring pond water microbes to focus under the microscope, which objective should you use?
4x
How will we attach bacteria to the glass before Gram Stain?
Heat fixed smear
What is the name of the counter stain that will turn Gram negative bacteria pink?
Safranin
What is a differential Stain?
It's a stain that renders one type of microbe one color and other types of microbes another color.
Challenge step
Rinse with acetone. This dehydrates and tightens the cell wall of Gram + (peptidoglycan) so rinse does not enter cell and remains blue black because of the Crystal Violet - iodine complex. Gram - allows the rinse through its Lipid was calling for a counter stain - Safranin
What are the two way to determine if an organism is alive?
Movement
Name two major representatives of microbes?
Gram +.-
Bacteria are the size mitochondria, an internal ..........of eukaroyotes
organelles
Where can you find squamous epithelial cells?
in side the mouth
Spell the first person's name to find microbes under microscope
Leeuwenhoek
Quiz 2
...
What color should a Gram + bacteria be if Gram stained?
blue purple black
What color should a Gram + bacteria be if it has simple stain?
blue (question)
The success of the Gram stain relies on the integrity of the cell .........
wall
Simple stains
Simple stains are + charged (cations). Most proteins are - charged (anions). Ionic attraction keeps the stain attached to the cells so rinsing w/ H2O does not remove color
The purpose of heat fixing
1. Bacteria is killed
2. Bacteria is stuck to the slide
Name an alga or protozoa you saw last class?
Green Alage, , Paramecium, spirogyra, diatoms,
Why cant you stain spores with Gram Stain?
Cell walls of most endospores are impermeable. You have to use steam to heat endospores and allow the Malachite Green to penetrate spore coat. Cells stain red and the spores stain green
What kind of cell walls do acid-fast bacteria have?
mostly made of lipids. Waxes. (mycobacteria)
What kind of stain makes different cell parts different colors?
differential staining
Can you assume a clear area in a Gram stained slide is a spore?
No. could have been done incorrectly
While steaming a slide, what should you do if the stain begins to dry out?
put a wet paper over it
Dilutions
10 (-1)=
10(-2)=
10(-3)=
10(-4)=
10(-5)=
10(-6)=
1/10=0.1 (one tenth)
1/100=0.01 (one hundredths)
1/1,000=0.001 (one thousandths)
1/10,000=0.0001 (one ten thousandths)
1/100,000=0.00001 (one one hundred thousands)
1/1,000,000=0.000001 (one one millionths)
What is the dilution factor?
=final volume / aliquot (before dilution)
Is it possible that a colony could have arisen from two or more cells that stuck together?
It is possible that a colony could have arisen from two or more cells that stuck together. Thus a colony forming unit (CFU) may have originated from one or more cells.
How do you prove a pure culture?
by taking a sample from colony and re streaking on new plate. The new sample should be made up of the same microbe. You would have to proof by showing that all the colonies on restreak are identical and Gram staining these to demo all cells are identical
What temps do thermophiles prefer?
Psychrophiles?
Mesophiles
Philism?
+45degrees Celsius
-20degrees Celsius
20degrees C to 45degrees C
optimal growth temp
If an organism can only grow in the presence of oxygen?
no oxygen?
with or w/out oxygen
can tolerate oxygen?
Obligate aerobes
Obligate anaerobes
facultative anaerobes
aerotolerant fermenter
What is a positive oxidase test
the change in color of the bacteria to dark blue w/in seconds
What is a positive catalase test?
generation of bubbles around colony
If you put 1 mL of soil into 99 mL of diluent, what is the dilution?
1/1+99=1/100 or 1:100
What is an axenic culture?
pure culture
Is a viable titer an underestimate or an overestimate of bacteria in soil sample? Why?
underestimate. you are only able to see visible and the count would have to be an underestimate.
Categories used to describe colony morphology.
Shape (circular, irregular.etc)
Margin (entire, unulated)
elevation (flat, raised, convex)
size (small, med, large,etc)
texture (smooth or rough)
Appearance (Glistening, dull)
Pigmentation (NON =cream,tan) (Pig = purple, red, yellow
What is zone of inhibition
the area around which microbes wont grow around an antibiotic disc
Bactericidal
Bacteriostatic
-static = inhibit growth
-cide = kill
Listerine
Does not kill all bacteria
Brownian motion
the random motion of small particles suspended in a gas or liquid
What do you do if you can't measure the diameter of a zone of inhibition
measure the radius and multiply by 2
How do you know if a colony is alpha hemolytic?
Alpha hemolysis is a greenish discoloration that surrounds a bacterial colony growing on the agar. This type of hemolysis represents a partial decomposition of the hemoglobin of the red blood cells. Alpha hemolysis is characteristic of Streptococcus pneumonia and so can be used as a diagnostic feature in the identification of the bacterial strain.
How do you know if a colony is Beta hemolytic?
Beta hemolysis represents a complete breakdown of the hemoglobin of the red blood cells in the vicinity of a bacterial colony. There is a clearing of the agar around a colony. Beta hemolysis is characteristic of Streptococcus pyogenes and some strains ofStaphylococcus aureus.
What will happen in MSA if the bacteria produces acid
Thus only halophiles or
halotolerant bacteria can grow on this medium and it allows the differentiation of colonies that
can produce acids from mannitol because these will cause the medium surrounding the colony to
become yellow
What 2 tests will determine O2 use by bacteria
oxidase
catalase
What kind of bacteria can grow on Nutrient Agar but not MSA
Non halophiles
Why is MSA both selective and differential
Because it only allows for halophiles to grow there so it selects halophilic microbes and it will change color making it a differential. also it allows for differentiation of colonies that can produce acids from mannitol because these will cause the medium surrounding the colony to become yellow
the pH near bacterial colony on MSA is 7. what color is the agar/
red
which agar should you use to observe hemolytic reactions?
blood agar
why do the catalase test last/
because it will destroy the colony once the test is performed.
steps to correctly streak an agar plate.
1. take an inoculating loop and flame it.
2. choose a colony from the agar plate with the colonies
3. dip the loop in the colony and get a sample
4. take the sample and streak 1/3 of a new agar plate
5. flame the loop again and let cool.
6. rotate plate 60 degrees and begin a new zig zag streak.
7. flame loop again and rotate again making the final streak from the last part of streak number 2
what bacteria should grow in lactose broth tubes?
e.coli coliforms. gass will be present in durham tubes in side the test tubes forcing the durham tube to rise.
what bacteria do you test in asaparagine broth tubes.
Pseudomonas
pos. result for lactose broth
gas in durham tubes
positive test for asparagine broth
glow under uv light
what agar plate do you use to confirm growth of e.coli
emb agar plate = eosin methyl blue agar
what does MPN stand for
most probable number
You have a lactose broth tube with gas, one with out, and asparagine broth tube that glows. which should you inoculate on emb plate.
the lactose broth tube with gas because it shows signs of e.coli and emb plates are for the growth of e.coli you have a green sheen.
what is positive test on EMB plate
green sheen
what happens to phenol red lactose broth if there is acid production?
there will be gas and change of color
how do we dislodge bacteria from hamburger?
put meat in blender with distilled water. this will dislodge bacteria from meat
which medium will you use to isolate proteolytic bacteria/
skim milk agar
which broth do you check under a uv light for positive test?
asparagine broth. (Pseudomonas)
positive result for methyl red test
red
citrate test show ability of bacteria to grow what as sole source
carbon
what is the top layer of the blended hamburger?
lipids
what is brownian motion
brownian movements happens to any and all particles free in liquid environment. if there is no movement they may be attached to immobile object
How can brownian motion be distinguished from motility
Motility gets cells somewhere. In brownian movement the cells appear to just bounce around
what are the differences between procaryotic and eukaryotic flagella
eukaryotic flagella similar to cilia and possess many microtubules where as prokaryotic flagella is one microtubule
In wet mount preparations, is it possible to see eukaryotic flagella? prokaryotic flagella
eukaryotic yes
prokaryotic no
Does crystal clear pond water contain living bacteria? What about air? Your finger
everything non sterile has bacteria
1. If your organisms were all blue, what is the Gram reaction?
If your organisms were all pink, what is the Gram reaction?
blue is gram+
pink is gram -
If your organisms were a mixture of blue and pink, what is the Gram reaction? Why? How
can that be proven?
gram +. if during prep some of the cells were damaged the stain would be allowed to leak out and thus giving you a false -
What are the functions of each ingredient in the medium
they provide sugars, amino acids, nucleotides, vitamins
What is the buffer in Nutrient Agar? Why do media have buffers
the buffers are amino acids. counter acts the acids or bases produced
What is the function of the nitrogen source? Sulfur source? Phosphorus source
the nitrogen is used for the synth of amino acids
sulfur used also for amino acids