MCB2004C Lab Midterm

How many BSLs are there?

4

BSL 1

Work with microbes that do not cause disease in healthy individuals

BSL 2

Work with organisms that are associated with human disease but are not spread by aerosols

BSL 3

Work with organisms that cause serious human diseases that are transmitted by aerosols

BSL 4

Work with organisms that cause life-threatening diseases by aerosols or unknown modes or transportation

Steps for aseptic transfer

1. Sterilize the inoculation loop by flaming

2. Use the loop to obtain the sample to be transferred

3. Transfer the sample to the sterile medium to be inoculated

4. Re-sterilize the loop by flaming

Why is working aseptically important?

To transfer microbes from one culture to another without contaminating the original culture, the sterile medium to be inoculated, the work area, or yourself

Clinical applications of aseptic transfer

Clinical microbiology laboratory and "bedside inoculations"

How do you sterilize a loop with a Bunsen burner?

1. Beginning at the base of the loop, pass the wire through the very tip of the blue inner cone of the flame. The wire will glow bright orange

2. Pull the wire up and through the flame, working your way toward the loop

3. Finish by flaming the loop portion till it briefly glows orange. Remove from flame and allow to cool for a few seconds

Culture Resuspension

The culture tube should be held loosely between the thumb and middle finger. Your grip should be secure enough to keep from dropping the tube, but loose enough to allow the tube to move a little. Use the index finger of your opposite hand to "flick” the bottom of the tube. When done properly, this will produce a vortex in the media that will lift sedimented organisms off the bottom of the tube and disperse them throughout the media. This step is crucial when you are working with liquid cultures, and failure to follow this step can result in few to no micro-organisms being transferred to your new culture or slide

What is the four-zone streak?

Used to separate the species contained in a mixed culture of 2 or more microbial species

Four Zone Streak Procedure

1. A small sample of culture is placed onto the first zone of the agar plate. This would equal one loopful of bacteria from a broth culture, or a very small portion of bacterial colony from a solid culture media. It is very important with solid culture to not use more than the smallest possible amount of the solid culture, otherwise the initial concentration of bacteria is too high to dilute sufficiently

2. The initial portion of bacteria is spread out over a small zone on the edge of the plate. The objective is to spread out the initial sample of bacteria as evenly as possible in this zone. The streak lines should overlap with each other, and it is advisable to go over this area multiple times

3. Flame the loop. YOU WILL NOT TAKE ANY MORE SAMPLES OF YOUR INITIAL CULTURE. When you start to streak in zone 2, your loops needs to be sterile

4. A second zone is created by spreading the top third of zone 1 across zone 2, which will have a reduced density (CFU/ml) compared to zone 1. After the creation of zone 4, flame your loop again so that it is sterile before you work on zone 3

5. Use approximately one third of zone 2 to streak into zone 3. Once again, flame your loop before proceeding to zone 4

6. Zone 4 is where full isolation of colonies is expected. Your streak pattern will not be as close together as the previous zones, but still fairly tight. You will also not cross back into zone 3 as often. Be absolutely certain that you do not cross into the first two zones at any time, ast this will ruin your chances at achieving good isolation

What is a CFU?

Colony forming unit. Due to the possibility of more than one cell of the same species establishing a colony when falling off the loop and onto the agar

Objective Lens

Located on the microscope’s turret (revolving nose piece) with 4 different magnifications (4x, 10x, 40x, 100x)

Ocular Lens

In the eye pieces of the microscope and are usually a magnification of 10x

Magnification

the process of visually enlarging an object's appearance using a dual-lens system

Resolution

Clarity of an image

For a well-adjusted bright-field microscope using a green or blue light source: 0.2 µm

What are wet mounts?

Used by clinical microbiology labs to rapidly diagnose superficial yeast infections caused by species of Candida and urogenital infections caused by the protozoan Trichomonas vaginalis.

What organisms could you use a wet mount for?

Candida and urogenital infections caused by the protozoan Trichomonas vaginalis.

What can simple stains tell you?

Morphology, size, and cell arrangement of a patient’s isolate that needs to be determined quickly, without having to perform a regular Gram stain

How are simple stains prepared?

1. Obtain a loopful of the suspension to be examined and place it on the slide

2. Spread out the drop so that it covers an area about the size of a nickel, and allow it to air dry (you should see a slight haze after it dries)

3. Pass the dried smear through the upper flame several times to heat-fix the smear. Do not over-flame

4. Place the heat-fixed smear on the support bars over the bench sink

5. Cover the smear with the methylene blue stain for one minute

6. Rinse the slide with tap water. Do this by tilting the slide at a 45 degree angle and gently squirting DI water at a spot slight above the smear, letting the water run over the smear and carry the excess dye off the end of the slide

7. Gently dry the slide with the bibulous pad

8. Observe the smear at 1000x total magnification using the oil immersion objective

Differential Stains

Staining procedures that require the use of more than one reagent to obtain the desired staining effect

What can differential stains tell you?

In addition to allowing the observation of cell shape, size, and arrangement, they can determine certain biochemical and structural properties of the cell

Gram Stain

Helps show Gram-negative (pink color, thin peptidoglycan layer, and significant amounts of lipids. EX: E. coli) and Gram-positive (purple color, thick peptidoglycan layer, and very little lipids. EX: Staphylococcus)

Reagents used in a gram stain and their purpose

Crystal Violet: Primary Stain

Gram’s Iodine: Mordant (traps dye)

95% Ethyl Alcohol: Decolorizer

Safranin: Counterstain

Gram-stain Process

1. Place the heat-fixed smear on the support bars over the bench sink

2. Stain the smear with the crystal violet stain for 1 minute

3. Tilt the slide and gently rinse the slide with tap water

4. Overlay the smear with Gram’s Iodine for 1 minute

5. Decolorize with 95% ethanol by tilting the slide and running the ethanol over the smear until the drops of ethanol running of the slide are clear. DO NOT DECOLORIZE FOR MORE THAN 10 SECONDS

6. Tilt the slide and gently rinse the slide with tap water. You must rinse the slide IMMEDIATELY after decolorizing. Do not let the alcohol sit on the slide or you will remove stain from the Gram-positive bacteria, giving you a false Gram-negative result

7. Counterstain by covering the smear with Safranin for 30 seconds

8. Tilt the slide and gently rinse with tap water

9. Gently blot dry with bibulous paper

10. Observe the smear at 1000x total magnification using the oil immersion objective (100x)

Common errors in a gram stain and what happens if you mess up a certain step?

1. Over-decolorization

   1. Causes Gram-positives to appear Gram-negative

2. Under-colorization

   1. Causes crystal violet to be retained in a Gram-negative cell when it shouldn't

3. The bacterium

   1. Gram stains performed on growth older than 24 hours causing aberrant stain results and sometimes over-colorization

What do gram stains tell you about a cell?

Can indicate the tentative identity and cell wall type of the infecting bacterium, providing important information for selecting an appropriate antimicrobial to treat the infection

What do acid-fast stains detect?

The presence of wax-like, high-molecular-weight fatty acids, such as mycolic acids, found in the cell walls of certain bacteria

What reagents are used in an acid-fast stain?

1. Carbolfuchsin: Stains cell purplish-red

2. Acid-Alcohol: Decolorizer

3. Methylene Blue: Counterstain

What genus would an acid-fast stain be used for?

Mycobacterium tuberculosis

What do positive results look like in acid-fast stain?

Purplish-Red

What do negative results look like in acid-fast stain?

Blue

When would you use random sampling?

When not looking for a particular species

When would you use direct sampling?

When looking for a particular species

Specific organisms you might detect with direct sampling

Legionella pneumophila on the shower heads in a patient’s bathroom

How are throat culture obtained?

1. Put on gloves and unwrap the tongue depressor and swab, being careful not to contaminate them by touching nonsterile surfaces

2. Insert the tongue depressor into the patient’s mouth, using it to hold down the rear of the patient’s tongue to gain access to the pharynx

3. Insert the swab into the patient’s mouth and gently swab the back of the throat, tonsils, and any areas showing increased redness or pus formation, then remove the swab

4. Inoculate the swab to a blood agar plate by swabbing a dime-sized area of the first quadrant, then streak for isolation using a standard four-zone streak method

What are throat cultures plated on?

Trypticase soy agar (TSA) plate with 5% sheep RBCs

What are you detecting with throat cultures?

The presence of Streptococcus pyogenes, the most common etiology of bacterial pharyngitis (strep throat)

How can colony characteristics be helpful?

1. Alpha Hemolysis: Fading or Yellowing

2. Beta Hemolysis: Clearing of the agar color

3. Gamma Hemolysis: No reaction

Determine which organism is the cause of bacterial pharyngitis (strep throat) and what is just the normal microbiota or the oropharynx

What is primary isolation media?

Culture media used by the clinical microbiology laboratory to isolate microorganisms from patient specimens

What is primary isolation media used for?

To support the growth of nutritionally fastidious organisms

What does it mean for a media to be selective?

Designed to encourage the growth of certain microorganisms while inhibiting the growth of others.

What does it mean for a media to be differential?

Contain indicators that allow us to detect metabolic differences between microorganisms

Is blood agar selective, differential or both?

Differential because it can detect and differentiate the hemolysis patterns of streptococci

Is MacConkey agar selective, differential or both?

Both selective and differential.

It is selective for Gram-negative bacteria because it contains bile and the dye crystal violet, which both inhibit Gram-positive bacteria

It is differential because it can differential lactose-fermenting bacteria from non-lactose fermenters. Lactose positive can ferment lactose (acid production = deep bright pink, while Lactose negative cannot ferment lactose (no acid production = clear/light pink color)

Is mannitol salt agar selective, differential or both?

Both selective and differential

Selective is due to its high (7.5%) sodium chloride concentration, which staphylococci are able to tolerate.

Differential because S. aureus and S. saprophyticus are able to ferment the mannitol in the medium. These two species will appear yellow and all other staphylococci growing will appear red.

What is nutrient agar used for?

Allows for the growth of nearly every organism inoculated on it and for growth control

How is blood agar made?

By adding sheep red blood cells to trypticase soy agar (TSA)

Can blood agar be modified to make a different agar?

By adding the sheep red blood cells to the TSA when it is at a higher temperature, causing the RBCs to lyse and release their contents into the medium. The heat denatures the hemoglobin, changing the color of the agar from red to brown, hence the name, chocolate agar

Numerical Aperture (NA)

A number that describes the light-gathering ability of a lens and its resolving power. The higher the NA, the higher the resolution of the objective

What happens at each step of the Gram staining process?

1. The crystal violet enters easily into most Gram-positive and Gram-negative cells.

2. Gram's iodine, the mordant, complexes with the crystal violet making it difficult to remove from the cell.

3. Exposing the crystal violet-iodine mixture to the decolorizer, ethyl alcohol, will make the lipid cell walls of Gram-negative bacteria become porous and the CV-I complex will escape, making the cell appear transparent. Gram-positive cell walls are not affected by the decolorizer and the CV-I complex is retained.

4. The safranin counterstain is used to stain the Gram-negative cells that are transparent after losing the CV-I complex. They will appear pink.