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


When you first bring pond water microbes to focus under the microscope, which objective should you use?


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?


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?


Name two major representatives of microbes?

Gram +.-

Bacteria are the size mitochondria, an internal ..........of eukaroyotes


Where can you find squamous epithelial cells?

in side the mouth

Spell the first person's name to find microbes under microscope


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 .........


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

10 (-1)=

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?

+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


-static = inhibit growth
-cide = kill


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


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/


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.


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


citrate test show ability of bacteria to grow what as sole source


what is the top layer of the blended hamburger?


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

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