Microbiology Lab

Front 1

Lab #1

Back 1

Ubiquity Testing, Aseptic Technique and Bacterial Isolation Techniques

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What are the two ways of making microorganisms visible?

Back 2

1. microscopy in conjunction with staining

2. culturing to obtain large enough numbers or biomass of microbes that can be observed with the naked eye

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binary fission

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single bacterial cell multiplies many times to produce a mass of genetically identical bacteria

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colony

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a group of microbial cells growing on a solid surface that is visible to the naked eye

- the cells are considered pure/identical

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medium (media is plural)

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material used to grow microorganisms which supplies microbes with nutrients, minerals and water

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fastidious microbes

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specific vitamins or special growth factors are added to the media, these microbes have special nutrient requirements

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broth

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liquid medium

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agar

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solid media

-a solidifying agent added to the media

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solid media

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referred to as nutrient agar

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What can form in the broth where the microbes are growing?

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1. pellicle - membrane on the surface of fluid

2. individual particles dispersed throughout the tube

3. some sink to the bottom to form a sediment

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Growth characteristics can relate to the bacterium's oxygen requirements, explain?

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1. bacteria with strict requirement for oxygen grow near the top of the tube

2. bacteria that needs limited or no oxygen grow in the middle or bottom

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What property of agar allows us to use the microbial isolation technique called pour-plate technique

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the melting temperature of agar is different from its solidifying temperature. This mean we can put the microbes in the agar while it is still semi liquid

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When are broth tubes used?

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when we want a large number of cells for a single application

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agar deep

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test tube with nutrient agar used for growing microbes with low or no oxygen (anaerobes)

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agar slant

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test tube of nutrient agar solidifying at an angle so there is more surface area for growth

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agar plate

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petri plate with nutrient agar that provides largest space for growth

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Aseptic Technique protects you from what?

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1. protects cultures from being contaminated

2. protects you and environment from being contaminated with microbes

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pure cultures

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only one species of microbes

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Why do loops and needles need to be sterilized before and after they are used?

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to prevent contamination of experiment samples or environment

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What should you do to keep the cultures from becoming contaminated?

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flame the lip of the test tub after opening and before closing

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Examples of Aseptic Technique

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1. flame lip of test tube

2. sterilize loop and needle after each use

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Two techniques that use agar medium for isolating various species of bacteria from a mixed culture

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1. spread plating of serial dilution

2. streak plating

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What are the advantages and disadvantages of streak plating

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faster than serial dilution but it takes practice

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What is the main reason to perform serial dilution?

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to determine the number of bacterial cells present in the original sample

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serial dilution

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bacteria in the original sample are serially diluted by transferring 3 times a constant volume of nutrient broth which results in cells being less concentrated in each tube

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how do you determine the original number of cells?

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use the dilution factor times the number of cells you can count

test tube two is 1:10

test tube three is 1:100

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What is N referred to in an experiment and how will achieve that.

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N is the number of times an experiment is repeated

-the higher the n value the more statistically sound the final calculations

-we will average the results of the entire class

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Lab # 2 Part 1

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Care and Use of the Microscope

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What did Antone van Leeuwenhoek call the microbes he saw in in microscope?

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Animacules

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Light Microscopes

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aka bright-field microscopes

-the specimen is a dark image on a field of bright light

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compound microscope

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the one we use in class refered to as bionocular microscopes

-two-lens system to magnify

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ocular lens

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the lens you look through aka the eyepiece

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objective lens

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the lens closest to the speimen

-has a higher capacity for magnification

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monocular microscope

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one ocular lens

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mechanical stage

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the surface where the specimen is sits

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mechanical stage adjustment knob

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moves the specimen right or left up or down

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Iris diaphrame

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adjusts the amount of light that hits the speciment and condenser lens

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total magnification of the image formula

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Total magnification = ocular lens magnification x objective lense magnification

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oil immersion lens

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designed for the lens to be dropped in so it can prevent refraction of light

-never use oil with any other lens

-clean after every use

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The the ways light can be adjusted are:

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1. using the rheostate or dimmer switch
2. adjusting the height of the condenser using the condenser height adjustment knob
3. opening and closing the iris diaphragm with the diaphragm adjustment lever

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Parfocal

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microscope property which keeps image in focus when changing from one lens to another

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working distance

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the distance between the specimen and the objective lens and when the lens is focused on the object

-oil immersion lens has the shortest working distance

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Resolution or resolving power (RP)

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the smallest distance between two nearby objects that can be clearly distinguished and also indicates the diameter of the smallest object

-clarity of a microscope

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Why is it important to keep the lens of microscopes clean before and after use?

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dust causes a decrease in resolution

-clean only with lint-free lens paper

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numerical aperture

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refraction of light of light results in decreased resolution

-the higher the NA value the better the resolution

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interpupillary distance

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the distance between the pupils and of the eyes

-adjust this to match your interpupillary distance

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ocular focus adjustment

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the ocular lens is focused independently
because your eye can't do it

-make this adjust to avoid closing one eye

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how does staining make microorganisms easier to see?

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adds color and contract to the cells

-to allow visualization of specific cellular structures

-shows shape, size and cell arrangement

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what are the disadvantages to staining?

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1. a long process to prepare

2. the microbes die

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There are 7 types of staining techniques:

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1. Simple stain

2. Gram-Stain

3. Acid-fast stain

4. Capsule stain

5. Endospore stain

6. Flagella Stain

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Simple Stain

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determines cell morphology, size and arrangement

-cytoplasm is stained

-negative stain - the background rather than the cells are stained

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Differential Stain

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distinguishes between two major groups of bacteria (gram+ and gram-) based on cell wall composition

-The Gram Stain

-The Acid-Fast stain

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The Gram Stain

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most important developed in 1884 by Han Christian Gram - the universal basis of classification and identification of bacteria

-either gram+ or gram- depending on composition of cell wall

-also useful for diagnosis, prognosis and selection of medical treatment

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The Acid-fast stain

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distiguishes mycobacteria from other bacterial genera

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Capsule stain (structural)

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determines whether the bacteria have a capsule of slime layer

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Endospore stain (structural)

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determines whether the bacteria produce spores

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Flagella stain (structural)

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determines whether the bacteria have flagella and are therefore motile