Midterm exam

What types of PPE are used in the lab?

Aprons, splash- and impact-resistant safety goggles (Z87), and gloves. If possible, also add a lab coat for safety.

What are fume hoods

They provide an enclosed air barrier between you and the hazardous materials used. They direct airflow away from you and carry those harmful contaminants out through the exhaust duct.

Where does trash go

Trash canister

Where do points go

Sharps container including razors and scapples

Where does glass go

Glass disposal

How is biological waste handled?

It goes in the biohazard’s container

How are chemicals handled

Go in hazardous waste containers

What is BSL?

Biosafety Level is the classification of microorganisms based on their:

• Potential to cause disease

• Conditions under which they should be handled

What does the CDC take into account when grouping a microorganism into a level

• Virulence

• Pathogenicity

• Antibiotic resistance

• Treatment availability

What level of BSL organisms are used in the lab?

BSL-1 and BSL-2

Why is the level of contamination measured as the number of colonies rather than the size of colonies

Their ability to contaminate is measured by their ability to multiply, as colonies originate from the division of a single cell.

• The size of the colony may only help with species identification.

Should one be concerned to find bacteria on the skin

No, because that is our normal microbiota that helps train our immune system, inform, modify, maintain organs, and digest food

How can microbial levels be controlled on the skin, surface, and air

Aseptic techniques are used to control microbial levels

• Skin= Washing hands

• Surface= Using disinfectants

• Air= Using adequate ventilation and airborne precautions

Why choose broth and gar plates

Agar plates are best for separating or isolating bacteria

Broth is convenient for growing large quantities of bacteria quickly

Why are agar plates incubated ‘upside down’ / inverted?

They are inverted so that condensed water does not drip into the agar, interfering with the growth of the colony

What is a colony?

A visible mass of cells resulting from the division of a single cell that can exceed one billion.

Review colony morphology / characteristics (shape, margin, and elevation).

Morphology is the form of organisms and their structures

Bacteria are described by their Configurations (round with scalloped margins, wrinkled, concentric, L-form, or Filamentous), Margins, and Elevation

Know basic use and care of a microscope.

Use both hands, one on the arm and one on the base of the microscope.

Limit of resolution of a typical light microscope VS unaided human eye

The limit of resolution of a microscope is 0.2 uhms, meaning it can distinguish 2 objects as distinct within this range. The unaided eye can only do this at 0.1 mm.

What 2 adjustments can be made to the condenser and how do they affect the image seen

The condenser can be moved up or down to change the focus of the light on the specimen. Up increases the light on the specimen, while moving it down decreases it.

What is a parfocal microscope?

The image remains both centered and in focus when changing from a low-power to a high-power lens

Parts of a light microscope

Remember!

• The condenser has a diaphragm that conducts AND regulates the amount of light to the specimen

• Objective lenses are:

  • Low-power, high dry, and oil immersion

What can be done to improve contrast and resolution when using a microscope?

Resolution: Ability to distinguish fine detail

Contrast: Difference in brightness between features

• Resolution can be improved by high NA, oil immersion, opening the diaphragm wider for higher resolving power, adjusting the condenser, and being aware of working distance

• For contrast, adjust the iris of the diaphragm (close down diaphragm) and use specialized optical techniques like phase contrast, dark field, or differential interference contrast

How to achieve the maximum resolution using oil immersion

Place immersion oil on the slide before using the lens

Open the iris diaphragm wider for higher resolving power

Adjust the condenser when changing objectives

Beware of working at a distance to avoid damage to the lens

How does oil work for oil immersion lens and not others

Oil immersion has the same refractive index as glass, so it creates a continuous lens system that prevents the loss of light when passing from glass to air

List 4 student errors that can result in a dirty or broken microscope.

• DO NOT use the coarse adjustment knob w/oil immersion lens

• Clean oil off the slide and oil immersion lens before switching lens

• Place the dust cover before storing it away

• DO NOT use only ONE hand to handle/transfer the microscope

What are aseptic techniques?

They are practices that ensure that

1. No microorganisms are introduced into culture materials

2. No contamination occurs to the handler

3. No contamination remains after work is done

List several basic steps one follows in accordance with aseptic techniques.

1. Use PPE (aprons, gloves, goggles, and a lab coat)

2. The work area is to be disinfected

3. Loops and needles are sterilized by a Bunsen burner

4. The mouth of the culture tube is flamed to be sterilized

3 reasons why aseptic technique is essential when handling microbial cultures

1. Ensures that only desired organisms are transferred in each inoculation

2. Kills microorganisms that may already be present in the workspace

3. Ensure bacteria deposited outside the workspace are killed, protecting us from infections

Provide 2 examples of how heat is used during inoculation of a tube culture

1. Used first on an inoculating loop to sterilize and remove undesired organisms

2. Used last on the inoculating loop to destroy remaining organisms on the loop

How is air contamination prevented when an inoculating loop is used to introduce or take a bacterial sample to/from an agar plate

The plate cover is held diagonally over the plate to allow the inoculating loop to gather bacteria w/o exposing the microbes to other bacteria for long

Where should a label be written on an agar plate

On the bottom of the plate or where the agar is present to ensure identification of the culture

How should agar plates be incubated and why?

They are to be incubated upside down (agar on top) to prevent moisture from condensing on the agar surface and spreading on the inoculated organisms

What are disinfectants good for? What organisms may still remain on the bench after disinfection?

They kill vegetative cells and viruses, but not endospores. Yet, they reduce the likelihood of contamination

How different is bacterial growth in broths, slants, and agar plates. Advantages and disadvantages of them?

Broths → Appears as a presence of cloudiness. Allows for a large presence of bacteria

Agar plates → Appears as visible, isolated colonies. Allows identification of different bacterial colonies

Slants→ Appears as visible, isolated colonies. Allow space saving environment for longer storage

How is a streak plate done?

An agar plate is divided into 4 areas, and one area is inoculated with a certain bacterium. The second area is then inoculated with a little bit of the bacteria inoculated in area 1. The same is repeated in areas 3 and 4 until the cells are diluted over the plate and single cells are separated and able to grow as isolated bacterial colonies.

Advantage of streak plate method over pour plate method

It is very quick to do and requires few materials, but it requires a certain level of skill

Advantage of pour plate method

Requires less skill than the streak plate method, but more materials

3 common errors during streak plating that may result in no isolated colonies after incubation

1. Failure to let the agar cool down if liquefied, to avoid condensation and moisture

2. Not inverting the plate to prevent condensation and moisture from reaching the agar plate

3. Forgetting to flame the mouth of the tube to destroy bacteria around it

You observe isolated colonies on quadrant 2 rather than 4. Why did this happen and would the streak plate be considered a failure

Yes, it is a failure bc the dilution process or spread of bacteria happened too soon, and thus proper isolation of pure culture did not occur

Difference in appearance of surface and subsurface colonies in a pour plate. If they are the same bacteria, why do these differences happen?

Surface colonies are larger in size than subsurface colonies. This is because cells were inoculated into the melted agar before the plate was poured

What is the significance of a streak plate?

It is a method used to obtain pure cultures that isolate a single kind of organism. This helps to study the organism’s culture, morphology, and physiological characteristics.

• It can also be used to determine the best treatment options

What are the basic steps in making a smear from solid media and from liquid media?

From liquid medium: 2+ loopfuls of the liquid medium are placed directly on the slide

From a solid medium: 2+ loopfuls of WATER are placed directly on the slide. AN inoculating loop then disperses the organisms in the water

What makes a good smear?

A good smear is done when cells are adhered to the slide of the microscope, shrinkage of the cells has not occurred, and a thin smear is done.

Why is it important to limit the quantity of cells used to prepare a smear

Thick smears w/large clumps can obscure details about arrangement and the presence of internal structures. It may cause staining to prevail in the clumps, preventing visualization

What is the purpose of heat fixation? What problems can arise when the slide is heated in a flame before it is completely dried out? What if you forget to heat fix before staining?

Heat is used to both kill and fix organisms on the slide

If done before the slide is dried, boiling and shrinkage of cells may occur, distorting morphology

If not done before staining, the bacterium may wash out during the rinse-out process of staining

What happens if you forget to turn the slide over after making a target circle and you apply the organism to the slide? After staining, the slide is placed right-side up. What problem will I face when trying to view the organisms?

I wouldn’t be able to focus on the microbes because the glass slide acts as a barrier, preventing sharp focus and clear viewing of details. Lenses may become dirty or contaminated as well, causing damage to the microscope and posing a hazard to me.

What is a chromophore?

Thus, they are attracted to bacterial cells with a negative charge on their surface.

What is the difference between a basic and an acidic dye?

Basic dyes have POSITIVELY charged (cationic)chromophores.

Acidic dyes have NEGATIVELY charged (anionic) chromophores.

Since basic dyes are positively charged, they are attracted to bacterial cells w/a negative charged surface and can stain cells

Acidic dyes are negative and repel from bacterial cells; thus, they only stain the background of the slide

Bacteria are usually stained with a basic dye – why?

Basic dyes can stain the bacterial cells as they have an electrostatic attraction between the negatively charged cells and positively charged chromophores of basic dye

What type of dye is crystal violet and what color do cells take on after it is applied?

It is a basic dye that stains deep purple

What is meant by the palisade arrangement of cells

Parallel arrangement of rod-shaped cells AKA “picket-fence”

What is expected to be seen if a Streptococcus pyogenes smear is stained w/crystal violet

Sphere-shaped cells arranged in chains, stained deep purple are to be expected

What does simple staining reveal

Morphology of the cells like their shape and arrangements

If you were working with an unlabeled simple-stained smear, would you be able to identify the bacterial species by observing it under the microscope?

Simple staining can reveal the shape and arrangement, but the specific bacteria won’t be identified because many microorganisms share these characteristics, so further investigation would have to be done.

What is the difference between a simple and a differential stain?

Simple staining → Reveals morphology (shape and arrangement of cells) using 1 dye

Differential staining → Uses multiple dyes to distinguish between different types of cells and their structures

Gram stain

Purpose: To differentiate between Gram-positive and Gram-negative bacteria based on their cell wall structure and composition

Results: Gram-positive takes on a purple color, and gram-negative takes on a red color

Stains: Crystal violet and Safranin

Procedures: After heat-fixing the cells, they are dyed with crystal violet. Gram’s Iodine (mordant) is added to create an insoluble complex in Gram-positive bacteria. Ethyl alcohol is used for decolorization. Safranin is then added as a counterstain.

Why is gram stain considered a differential stain

It uses 2 kinds of dyes to differentiate between 2 kinds of cells based on their different staining reactions that structures have w/different dyes.

How does age of a culture affect gram staining reactions

The optimum age is 16-8 hours, as anything beyond is when cell walls begin to break, causing them to be leaky and lose crystal violet.

What step in gram staining is more prone to error? If done right, how might it affect the end result?

Decolorization is the most critical step bc if it

Negative stain

Purpose: Negative dyes are acidic, so they reveal the morphology of cells, such as external structures like capsules, by staining the background.

Results: Cells appear as clear objects against a dark background

Stains used: India Ink or Nigrosin

Procedure: Organisms are mixed in a drop of nigrosin and spread over the slide by another slide, making a thin-to-thick smear.

Another is the needle method that just spreads nigrosin and the bacteria w/o the need for a slide.

What type of chromophore is associated w/a negative stain

Negative stains are acidic and have negatively charged chromophores that do not penetrate the cell, thus only stain the background or surrounding of the cell

What step normally associated w/staining bacterial cells is omitted when the size of the cell is determined?

Heat-fixation is avoided to prevent the shrinkage of cells and accurately determine the size of bacterial cells without harming the cells’ structure

Acid-fast stain

Spore stain

Review the Disk Diffusion test (Kirby Bauer method) – review the questions on the lab reports.

What are the standard procedures / protocols required for setting up the Kirby Bauer method? List 4.

What is a zone of inhibition?

Know the mode of action of the following antimicrobials: Penicillin, Imipenem, Tetracycline, Ciprofloxacin, Streptomycin, and Sulfisoxazole-Trimethoprim (folic acid inhibitor – a competitive inhibitor), Isoniazid.

Note the difference between antiseptic and disinfectant.

Review questions in lab report (Ex 32)

Review the effects of UV as a control agent – review the questions on the worksheet.