Microbiology Lab Exam 1

Conversion of Millimeters to Microns

1 millimeter= 1000 micron

What reagents are used in a capsule stain?

1. Congo Red #1 2. Congo Red #2

Gram Staining Steps

1. Crystal Violet rinse 2. Gram's Iodine rinse 3. Ethyl Alcohol rinse 4. Safranin rinse

How do you transfer from slant to slant?

1. Heat inoculating loop 2. Flame tube mouth 3. Pick up organism from slant with loop 4. Flame tube mouth 5. Recap slant culture 6. Flame tube mouth of sterile slant 7. Slant surface streak with unflamed loop in a zigzag motion 8. Flame tube mouth and loop

How do you transfer from broth culture to broth culture?

1. Heat inoculating loop 2. Shake tube to disperse organisms 3. Tube lid removed and mouth of tube flamed 4. Loopful of organisms removed from tube 5. Loop removed from culture and tube flamed 6. Cap removed from sterile broth and cooled loop is inserted into sterile broth 7. Loop and mouth of tube are both flamed

How do you transfer from broth to plate?

1. Heat loop and mouth of culture broth tube 2. Remove loopful of organisms from culture broth using cooled inoculating loop 3. Gently streak loop across plate 4. Lid plate and flame loop and mouth of tube

Steps for Capsular Stain

1. One drop of Congo Red #1 to edge of one slide 2. Transfer loopful of K. Pneumonieae to drop and mix 3. Spread into thin smear with a second slide and let air dry 4. Stain with Congo Red #2 and rinse with water

How do you prepare a bacterial smear?

1. Place two or more loopfuls of liquid medium directly on the slide (if from broth) 2. Place one or two loopfuls of water on slide and then use an inoculating loop to disperse the organisms in the water (if from solid media) 3. Spread the organisms over the area 4. Allow slide to air dry 5. Heat fix the slide (Make sure smear is thin)

Steps for Spore Stain

1. Prepare smear of B. Megaterium by mixing small amount with water 2. Add loopful of S. Epidermis to same drop and heat fix 3. Add small piece of paper towel over your dry smear and place slide over can that is heated over boiling water 4. Add malachite green to paper towel and keep it saturated 5. Rinse with distilled water 6. Safranin 7. Rinse 8. Blot Slide

4 Objective Lens Names and Magnification

1. Scanning Objective- 4x Magnification 2. Low Power Objective- 10x Magnification 3. High Dry Objective- 40x Magnification 4. Oil Immersion Objective- 100x Magnification

What are the main reasons for using aseptic technique?

1. To ensure no contaminating microorganisms are introduced into cultures or culture materials 2. No ensure the microbiologist is not contaminated by cultures that are being manipulated 3. Ensures no contamination remains after working with cultures 4. Reduce likelihood of contamination

What is a pure culture?

A culture in which contains only one single kind of organism

What indicates a successful capsular stain?

A halo around the cell with a dark background

Streak plate method

A method used to obtain pure cultures in which the inoculum is streaked over the agar in such a way that it "thins out," until a single cell is deposited in an area and grows to produce an isolated bacterial colony

Colony

A visible mass of organisms all originating from a single mother cell, a clone of bacteria all genetically alike

What does the capsule stain allow us to see?

An extracellular gel-like layer outside of the cell wall that is called a capsule

Organisms utilized in spore stain

Bacillus Megaterium: Bacillus (rod shaped), Endospore forming (Green) Staphylococcus Epidermid: Coccus (Circles), not endospore forming (Red)

Which genera form endospores?

Bacillus and Clostridium

Transient Bacteria

Bacteria temporarily living in or on a certain body part, such as the hands, removed with relative ease.

Coarse Adjustment

Brings the specimen into general focus, focuses the specimen under lower power

How do you sterilize an inoculating loop or needle?

By inserting it into a Bunsen burner until it is red-hot. This will incinerate any contaminating organisms that are present. Allow it to cool before picking up inoclulum.

Objective

Cylinder containing lense that collects light and enhances magnification

What is the most critical step of the Gram stain procedure?

Decolorization - If it is overapplied the dye-mordant complex may be removed from gram-positive cells, causing them to incorrectly appear as gram-negative cells

What is the name of the slide used in the hanging drop method?

Depression slide

What is the proper location for bacterially contaminated materials?

Designated waste collection are in the southeast corner

Purpose of endospore stain

Differential stain used to visualize bacterial endospores. They are not easily penetrated by stains so basic stains such as Crystal Violet will not penetrate.

Why is the gram stain considered a differential stain?

Differential stains take advantage of the fact that structures within cells display different staining reactions that can be distinguished by the use of different dyes. In the Gram stain, gram-positive and gram-negative cells are differentiated based and their cell wall structure and composition. They can be identified by their colors after performing the stain.

What 3 organisms normally live on human skin?

Diptheroids Staphylococci Fungi

What is an endospore?

Dormant structures that can survive environmental conditions that are not favorable for bacterial growth.

What color is the endospore in the spore stain?

Green

Gram- Positive

Have a thick layer of peptidoglycan that comprises the cell wall of the organism outside of the cell membrane - Appears purple after the staining procedure

What is the mordant in the spore stain?

Heat

Explain why plates should be inverted during incubation

If the plate is not incubated in an inverted position, condensation from the dish lid will be deposited on the agar surface, dispersing the organisms, disrupting the desired growth pattern, and preventing the formation of individual colonies.

What is the importance of the Gram stain in microbiology?

It is a valuable diagnostic tool that is used in the clinical and research setting. It is often the first test conducted on an unknown species in the lab and can provide presumptive identification of an unknown organism. It may be used to determine an appropriate treatment for a bacterial infection in a clinical setting.

Why is hand hygiene important?

It is the most important technique in preventing and controlling the transmission of infection. It is an effective way to remove opportunistic pathogens that occur on the skin.

Organism utilized in Capsular Stain

K. Pneumonieae: Bacillus (rod-shaped), Cell (Dark Purple), Capsule (white halo)

Illuminator

Light source for the microscope

Fine Adjustment

Moves the stage slightly to sharpen the image, sharpens the image under high magnification

What kind of plates are used in the Kirby- Bauer method?

Mueller-Hinton

How to measure an organism

Multiply the number of lines the organism covers by the calibrated distance for that objective

Are capsular stains heat-fixed?

No, heat fixing will destroy the capsule

Resident Bacteria

Normal microbiota on a body that are more difficult to remove because they reside in hair follicles or are entrenched in the skin.

Organisms utilized in Gram Stain

Pseudomonas Aeruginosa: Should appear pink/red rods (Gram Negative) Staphylococcus Aureus: Should appear purple cocci (circles) (Gram Positive)

What color is the vegetative cell in the spore stain?

Red

Gram- Negative

Sandwiched cell membrane around a thin layer of peptidoglycan - Appears pink/red after staining

How to calibrate ocular micrometer

Stage Count/Ocular Count x 0.01mm x 1000

Why is steam used in the spore stain?

Steam is used to more readily penetrate and make the stain become more entrapped in the endospore.

Stage

Supports the slide being viewed

Why is it important to remove tape from tubes?

The tape will adhere to the tube in the autoclave if left on

Zone of Inhibition

The zone around the saturated disk where there is no growth due to the agent inhibiting or killing the organism

Why do you wipe down the bench top before and after lab?

To get rid of any microorganisms

Why do you leave your lab coat in lab?

To protect yourself from contamination, since the lab coats are contaminated they should not be worn outside of the lab

How do you disinfect your area?

Treated with a disinfectant and spread with a sponge and left to air dry to kill any microorganisms that may be present.

How do you focus on a specimen?

Using the coarse/fine adjustment knobs as well as the condenser and illuminator to adjust the amount of light coming through

What is the purpose of immersion oil?

When placed between the objective and the specimen the oil forms a continuous lens system that limits the loss of light due to refraction. This reduces light refraction and maximized the numerical aperture to improve the resolution. Used under oil immersion objective which is 100x magnification.

Oculars

eyepiece lenses (usually 10x magnification)

How to calculate total magnification

power of objective x power of eyepiece (10x) ex.) oil immersion objective =100x total Magnification (10x100)

What conditions trigger endospore formation?

unfavorable conditions for bacterial growth - ex) lack of nutrients

Characteristics of endospores

- Dehydrated and not actively metabolizing - Resistant to heat (associated with water content), radiation, acids, and many chemicals ( due to the fact they have a protein coat that forms a protective barrier around spore) - Calcium dipicolinate and spore-specific proteins form cytoplasmic gel that reduces protoplasmic volume of endospore to a minimum - Thick cortex formed and contraction of it results in smaller dehydrated structure - Control amount of water entering endospore so can maintain dehydrated state

What two method were used to determine motility?

- Hanging drop slide - Tube method

What does MRSA stand for? What % of organisms are this type?

- MRSA: methicillin- resistant s. aureus - Less than 2%

Which organism is motile and non-motile in the experiment?

- Proteus Vulgaris: Motile - Micrococcus Luteus: Non-motile

How do you differentiate motile from non-motile organisms?

- look for directional movement that is several times the long dimension of the bacterium - If organisms are motile they will swim away from the line of inoculation into the surrounding medium, this will cause the medium to look cloudy, or turbid. Non-motile organisms will only grow along the line of inoculation