Lab - Mid-Term (1-8)

What microscope is used in the virtual lab?

A light microscope, specifically an oil immersion microscope.

How does the oil immersion microscope magnify images?

It uses a series of lenses to magnify the sample on the slide.

What is the function of the diaphragm in a microscope?

The diaphragm adjusts to let more light through.

What part of the microscope moves the stage up and down?

The coarse adjustment moves the stage up and down.

When focusing a microscope, which adjustment should you always start with?

You should always start with the coarse adjustment.

Which lens should you begin with when viewing a sample?

You should begin with the 10x objective lens.

Why is the oil immersion lens used?

The oil prevents light distortion and helps make the image clearer.

How many drops of oil should be used for the oil immersion lens?

You should just use one drop of oil.

What color indicates gram positive bacteria?

Gram positive bacteria is purple.

What color indicates gram negative bacteria?

Gram negative bacteria shows up pink.

Name two gram negative bacteria associated with contaminated food.

E. Coli and Salmonella.

What process ensures milk and yogurt are safe for consumption?

The bacteria in milk and yogurt are pasteurized, killing the bacteria.

How should you clean the oil immersion lens?

Clean it with a piece of lens paper to wipe the excess oil.

What sterilizes equipment with high pressure steam?

The autoclave sterilizes the equipment.

Were the yogurt samples contaminated?

Yes, they were contaminated.

What could happen to yogurt that was solid or disturbed?

The plant operator might have to recall any yogurt that was distributed or sold.

Calculate the total magnification for the 10X ocular lens with the 100X objective lens.

Total magnification is 1000X.

What is the first step to start the Gram Stain Technique lab in BIOL 221?

Read through the 'Module Instructions Gram Stain' and click 'Start' to begin.

What does the Gram Stain Technique help identify in bacteria?

It helps identify whether bacteria are Gram Positive or Negative and their shape.

What is the scientific name format used to identify bacteria?

The scientific name is given as Genus and Species.

In the virtual lab, how many slides are analyzed for Gram staining in BIOL 221?

There are a total of 8 slides analyzed in Microbiology Labs.

What tool is used to complete the Gram Stain assignment in the virtual lab?

The 'View Slide under the Microscope' option is used to complete the assignment.

How do you classify bacteria after analyzing the slides?

Bacteria are classified as Gram Positive or Negative, their shape identified, and scientific names recorded.

What is the first step in the Gram stain procedure after preparing the slide with bacteria?

Heat fix the slide by passing it through the flame of a Bunsen burner for 1-2 seconds.

What is the purpose of flooding the slide with crystal violet in the Gram stain procedure?

Crystal violet is the primary stain that will color all cells.

What happens if the slide is over-decolorized during the Gram staining process?

Gram-positive bacteria will appear gram-negative.

What is the role of iodine in the Gram staining process?

Iodine acts as a mordant, helping to fix the crystal violet stain in the cells.

What should you do if you observe that your slide contains neither E. coli nor Staph. aureus?

Review the steps and repeat the exercise, considering possible mistakes.

What can occur if your smear is too thick before staining?

Cells in very thick areas will not decolorize properly, and gram-negative bacteria may appear gram-positive.

What is the final step after adding safranin to the slide?

Rinse the slide with water and then view it under the microscope.

What could happen if the slide is not heat-fixed properly?

The smear may wash off during rinsing.

How long should the slide be flooded with safranin during the Gram staining process?

For 1 minute.

What can insufficient time for safranin counterstain result in?

Gram-negative bacteria may appear very faint and difficult to see.

What is the first step done to identify organisms in a stool sample from a patient with severe diarrhea?

The first step is to perform a Gram stain on the sample.

What happens if the smear prepared for Gram staining is too dense?

If the smear is too dense, it may hinder the staining process and lead to inaccurate results.

What is gram variability and how can it affect results?

Gram variability refers to the ability of some bacteria to stain differently due to changes in their cell wall structure; this can affect the reliability of the Gram stain results.

What are Gram indeterminate organisms?

Gram indeterminate organisms do not consistently show a specific Gram reaction and can appear as either Gram-positive or Gram-negative.

What is the advantage of Gram stain over simple stain?

The advantage is that Gram stain differentiates between Gram-positive and Gram-negative bacteria, providing more information about the organism.

What is the theory about the mechanism of the Gram-stain reaction?

The Gram-stain reaction mechanism involves differences in the peptidoglycan layer and outer membrane of bacterial cells, which affects how they retain the crystal violet stain.

Which step is the most crucial in Gram staining and why?

The decolorization step is the most crucial as it determines whether the bacteria will retain the primary stain or lose it to reveal the counterstain.

Why must you use young cultures for Gram staining?

Young cultures provide more accurate staining results, as older cultures may have altered cell walls which can affect the outcome.

What part of the bacterial cell is most involved in Gram staining and why?

The cell wall is most involved in Gram staining because its composition determines retention of the crystal violet stain.

What is the function of the iodine solution in Gram staining?

Iodine functions as a mordant, forming a complex with crystal violet that helps bacteria retain the stain; if omitted, bacteria might not retain the primary stain properly.

What is the purpose of the alcohol solution in the Gram stain?

The alcohol solution acts as a decolorizer, differentiating between Gram-positive and Gram-negative bacteria based on their cell wall structure.

What counterstain is used in Gram staining?

Safranin is the counterstain used; it is necessary to visualize Gram-negative bacteria after decolorization.

Could colors other than red be used for counterstaining?

Yes, although red is commonly used, other colors could theoretically work as long as they contrast with the primary stain.

What is the primary objective of the Gram stain technique in microbiology?

To differentiate between Gram positive and Gram negative bacteria.

Who discovered the Gram stain technique and in what year?

Hans Christian Joachim Gram in 1884.

What are the two major categories of bacteria differentiated by the Gram stain?

Gram positive and Gram negative.

What color do Gram positive bacteria stain?

Dark purple.

What color do Gram negative bacteria stain after the Gram stain procedure?

Red or pink.

What is the primary stain used in the Gram staining process?

Crystal Violet.

What step follows the application of the primary stain in the Gram stain procedure?

Addition of Gram's Iodine.

What happens during the decolorization step of the Gram staining process?

Alcohol or acetone dissolves the lipid outer membrane of Gram negative bacteria, resulting in them becoming colorless, while Gram positive bacteria remain purple.

What is the final step in the Gram stain procedure?

Counterstaining with Safranin.

Why is the Gram stain an important preliminary step in bacterial characterization?

It allows for the initial classification and identification of bacteria based on their cell wall characteristics.

What role does Lipoteichoic acid (LTA) play in Gram positive bacteria?

It has antigenic properties that stimulate immune responses upon cell death.

What is lipopolysaccharide (LPS) associated with in Gram negative bacteria?

It is a virulence factor that can elicit an innate immune response in humans.

What are the four basic steps of the Gram stain procedure?

1) Application of Crystal Violet, 2) Addition of Gram's Iodine, 3) Decolorization with alcohol, 4) Counterstaining with Safranin.

What type of bacteria is Staphylococcus aureus classified as after Gram staining?

Gram positive.

Can you name a common Gram negative bacterium?

Escherichia coli.

What is a typical mistake during the decolorization step of the Gram stain?

Over-decolorization can make Gram positive bacteria appear Gram negative.

How should microscopic slides be prepared for Gram staining?

Slides should be clean and free of grease or oil.

What is the purpose of heat fixing the bacterial smear?

To kill the bacteria, adhere the smear to the slide, and allow for better staining.

What is the typical size range of bacteria?

They are typically micrometers in size (1µm = 10-6 m).

What is the primary objective of aseptic technique in microbiology?

To prevent contamination of cultures and media from microbes in the environment.

What are the essential skills that microbiologists need to acquire through aseptic technique?

Transferring cultures, inoculating media, and isolating pure cultures.

Why is flaming the inoculating loop important?

Flaming the loop sterilizes it by killing all contaminating organisms.

What should be done to the loop before picking up organisms from the inoculum culture?

Allow the loop to cool slightly.

What happens if the inoculating loop is too hot when used?

It may create aerosols, which could introduce bacteria into the air and potentially into the respiratory tract.

What is the significance of flaming the mouth of the test tube?

It creates a convection current that helps prevent airborne contaminants from entering the tube.

What is the purpose of using a zigzag formation while inoculating an agar slant?

To lightly draw the loop over the surface without breaking the agar surface.

What does the streak plate method aim to achieve in microbiology?

Isolate individual microbial colonies from an inoculum by creating areas of increasing dilution on an agar plate.

What appearance indicates that the agar surface grows microorganisms that are genetically similar?

The culture is then considered as a pure culture.

What two colors denote Gram-positive and Gram-negative bacteria, respectively?

Gram-positive bacteria stain purple and Gram-negative bacteria stain pink.

What is the role of the mordant (Gram's iodine) in the Gram stain process?

It increases the affinity of the cell wall for the primary stain, Crystal Violet.

What bacterial morphology indicates Gram-positive cocci?

They may occur in pairs (diplococci), chains (Streptococci), or clusters (Staphylococci).

What would happen if the decolorization step during Gram staining is not timed correctly?

Over-decolorization can result in Gram-positive organisms appearing Gram-negative.

Why is it crucial to let the alcohol decolorizer flow slowly on the smear?

To avoid disrupting the smear while washing away excess stain.

What is the composition of the Gram-negative cell wall?

It has a thinner layer of peptidoglycan (10% of cell wall) and has an outer membrane containing lipids.

List two examples of Gram-positive bacteria mentioned in the notes.

Staphylococcus aureus and Streptococcus pyogenes.

List two examples of Gram-negative bacteria mentioned in the notes.

Escherichia coli and Salmonella typhi.

What does 'aseptic' mean in microbiological practices?

Without microorganisms.

What is the purpose of aseptic transfer techniques?

To protect the researcher from infectious agents and to protect pure cultures from contamination.

What should be done first when preparing for an aseptic technique?

Switch on the light and blower, then open the laminar airflow.

What color should the flame be when using a burner in aseptic technique?

Blue.

Describe the initial heating process for the inoculation loop.

Heat the inoculation loop with the burner horizontally from its base to vertical position until it becomes red hot.

What must you avoid doing with the inoculation loop after it has been cooled?

Never wave or touch the loop to the sterile agar surface.

What should you do after picking an isolated colony with a cooled loop?

Insert the loop into the sterile broth after heating the mouth of the test tube.

What is the appearance of the resulting solution after inoculation?

Yellowish in color.

How long should the test tube be kept in a shaking incubator?

Twenty four hours.

What is the resulting solution obtained from the inoculation process known as?

Inoculated broth culture.

Which of the following salt agars inhibit the growth of most gram-positive bacteria? EMB, Hektoen or all of above

Both EMB and Hektoen agars inhibit gram-positive bacteria due to their selective agents, allowing for the growth of gram-negative bacteria.

On EMB agar, ____ has a metallic green sheen appearance.

Escherichia coli

What are the two indicators for EMB?

Methylene blue and eosin Y

Mannitol salt agar are used for the genus …

Staphylococcus

Maconkey’s agar contains ___ that inhibits most gram positvie bacteria, except enterococcus and some species of staphylococcus? Cholesterol, lactose, sucrose, or bile salts?

bile salts, which prevent the growth of most gram-positive bacteria while allowing gram-negative bacteria to thrive.

True or false: the indicator in mannitol salt agar is neutral red.

False; the indicator is phenol red.

Hektoen enteric agar is selective and differential agar primarily used to differentiate ____ and ____.

Salmonella and Shigella

true or false: development of yellow halos in mannitol salt agar is evidence of pathogenic bacteria?

True; yellow halos indicate the fermentation of mannitol by pathogenic Staphylococcus species.