Lab pre-labs and reports 1-3

In VICI 325, what lens requires oil? Why do we need to use oil to view microbes?

100x lens. Because the microbes are too small to see with just the 100x lens. The oil refracts the light and gives the highest resolution.

Selective medium

7.5% NaCl agar

Select the INCORRECT statement A) Methylene Blue is a reagent used in simple stains B) A simple stain can be used to distinguish bacteria with a thick peptidoglycan layer from bacteria with a thin peptidoglycan layer C) A simple stain allows for determination of size and shape of the cells D) Gram's stain is a commonly used differential stain technique

A simple stain can be used to distinguish bacteria with a thick peptidoglycan layer from bacteria with a thin peptidoglycan layer

During a smear preparation, why is it necessary to spread out your sample to achieve a thin smear? A) A thin smear of bacteria allows for more accurate staining B) A thin smear provides a better chance to view individual cells C) There is no need to spread out a sample to create a thin smear D) A thin smear of bacteria allows for more accurate staining AND provides a better chance to view individual cells

A thin smear of bacteria allows for more accurate staining AND provides a better chance to view individual cells

Group A gram staining is purple Group B gram staining is pink Explain why

A. The bacteria is gram (+). It is a purple color because of the thick layer of peptidoglycan. The crystal violet and Gram's iodine will stain the thick layer. B. The Bacteria is gram (-). It is a pink color because it has a thinner layer of peptidoglycan under the outer membrane. Crystal violet is able to stain it, but is washed away easily by the acetone/ alcohol mixture because of the thin peptidoglycan layer. Only the safranin is able to stain the outer membrane layer.

What type of stain do we perform on Mycobacterium sp.? A) Gram stain B) Acid-fast stain (kinyoun or Ziehl-Neelsen) C) Mycobacterium sp. do not stain D) Capsule Stain

Acid-fast stain (kinyoun or Ziehl-Neelsen)

In this course we may see which of the following A) Non-hemolytic bacteria B) Bacteria that produce complete hemolysis C) Bacteria that produce incomplete hemolysis, although this type of hemolysis is likely to be very difficult to see D) Bacteria that produce double-zone hemolysis

All of them

Which of the following are true? A) A thin smear is best for bacterial staining because it will allow for proper staining of the cells B) A thin smear is best for bacterial staining because it will allow visualization of the true shape of the cells C) A bacterial smear should be air dried before heat fixing in order to avoid distorting the shape of the cells D) It is very important to heat fix your bacterial smear before performing a gram stain so that the cells do not wash off the slide during the procedure

All of them

While inoculating a broth from a broth, you should: A) Ensure bacteria are in suspension B) Sterilize your loop using the incinerator C) Utilize your pinky finger to hold the tube cap

All of them

Differential Medium Define

Allows us to differentiate between 2 or more organisms

In what order (from beginning to end) are the reagents used when performing a Gram stain procedure? A. Acetone/ Alcohol mixture B. Crystal violet C. Gram's iodine D. Safranin E. Acid/ Alcohol Mixture

B, C, A, D

Enriched growth medium

Blood agar

We use many forms of culture media in microbiology. Which of the following is NOT a type of culture media? A) Broth tube B) Broth plate C) Agar slant D) Agar plate

Broth plate

Once you have focused on your specimen using the 10x objective, the next step in setting up Kohler illumination is: A) Push the iris diaphragm completely to the left B) Close the field diaphragm by turning it counter clockwise C) Use the coarse focus knob to raise the stage to the top

Close the field diaphragm by turning it counter clockwise

The components of (reagents used in) the Gram Staining procedure are: A) Crystal violet, Gram Iodine, Acetone-alcohol or 95% alcohol decolorizer, and Safranin B) Crystal Violet and 20% Copper Sulfate C) Malachite Green and Safranin D) Carbol-fuchsin, Acid-alcohol, and Methylene Blue E)

Crystal violet, Gram Iodine, Acetone-alcohol or 95% alcohol decolorizer, and Safranin

In which step of the gram staining procedure is the timing most important and why?

Decolorization using acetone alcohol. Decolorize from 4-6 seconds, depending in which gives the best results. Too short of a time, and gram - bacteria may not be decolorized enough. Too long of a time, and gram (+) cells will also be decolorized.

In which type of media do we know the exact and complete composition? A) Enriched growth medium B) Selective medium C) Differential Medium D) Defined medium

Defined medium

In which type of hemolysis do two different enzymes diffuse from the bacteria colonies at different rates? A) Incomplete B) Complete C) Double zone D) No hemolysis

Double zone

Acid-Fast organisms, such as Mycobacterium spp. stain easily with a Gram Stain procedure

False

Capsules staining procedures include heat fixing your slide prior to staining True/ False

False

Endospores are easy to stain and are produced by a wide variety of bacteria True/ False

False

Once you have transferred your bacteria to a slide, you immediately heat fix the smear to adhere it to the slide True/ False

False

The decolorizor (alcohol), washes the crystal violet out of the thick peptidoglycan layer of gram (+) organisms True/ False

False

There should be a thick smear of bacteria on the slide to better see important features of the organisms True/ False

False

You can use the 40x objective lens (400x total magnification) to make useful observations of bacteria True/ False

False

In a correct Gram Stain A) Gram (+) bacteria stain pink B) Gram (-) bacteria stain purple C) Gram (-) bacteria stain pink D) Gram (+) bacteria do not stain any color

Gram (-) bacteria stain pink

For the microscopes in VICI 325, why do we perform "Kohler illumination"? What does this achieve?

It provides the best balance between contrast and resolution when looking at microorganisms by spreading the light evenly over the field of view

Defined medium

Known exact composition

Which statement is false regarding incomplete hemolysis A) It is also known as alpha hemolysis B) Methemoglobin degrades to hemoglobin, which results in the green color C) The hemolysins lyse red blood cells with no enzymatic effect on hemoglobin

Methemoglobin degrades to hemoglobin, which results in the green color

Regarding hemolysis, which statement is correct? A) Phospholipase enzymes create channels through a red blood cell's cytoplasmic membrane B) Porin hemolysins break down the phospholipids of red blood cell's cytoplasmic membranes C) Most hemolysins break down the phospholipids of cytoplasmic membranes in red blood cells D) None are correct.

Most hemolysins break down the phospholipids of cytoplasmic membranes in red blood cells

You are given an unknown bacteria. Dr. Turner asks you to determine if this bacteria have flagella. What type of medium should you use to determine this? A) Differential medium B) Motility test medium C) Defined medium D) Selective medium

Motility test medium

How do we find total magnification when using the compound microscopes?

Ocular lens magnification (10x) x objective magnification

Medium X has the characteristics: Favors the growth of particular microorganisms Specifically inhibits the growth of other microorganisms Contains 1/ more inhibitors What media type is Medium X? A) Selective B) General purpose C) Differential D) Selective/ Differential

Selective

Selective/ Differential medium

Selects for certain organisms and allows us to differentiate organisms that are capable of growth on the medium

If streak series 1 is made from the original bacterial culture, which of the following statements is TRUE? A) Streak series 3 has the most bacteria B) Streak series 2 has the most bacteria C) Streak series 1 will likely contain the most isolated colonies D) Streak series 3 will likely contain the most isolated colonies

Streak series 3 will likely contain the most isolated colonies

Explain the process and importance of streaking for isolation

Streaking for isolation starts off with series 1 being of the original bacterial culture. The loop is then sterilized for the 2nd series. The loop should also be sterilized after each streak. Streak 2 is then made perpendicular to the edge of streak 1, using bacteria from the previous series. Streak 3 is the same repeated process. Each series dilutes the bacteria from the previous streak, so there should be a lower concentration of bacteria in each series. The last series should have the most obvious isolated colonies.

Why should agar plates be kept inverted during incubation?

The agar plates should be inverted during incubation to prevent condensation from falling onto the bacteria and contaminating the growth. The water can also mix with the bacteria and mess with the individual colony growth.

You observe a bacteria with incomplete hemolysis. Which is true? A) The green color is created by enzymatic action that transforms hemoglobin to methemoglobin B) The green color is created by natural degradation of hemoglobin to methemoglobin C) The green color is often very easy to visualize D) None are true

The green color is created by natural degradation of hemoglobin to methemoglobin

We do not use Heat fixation when performing a capsule stain. Explain Why

The heat can destroy the capsules and cause the bacteria to shrink. It would then look as if the bacteria didn't have a capsule.

Select the incorrect statement regarding the motility test medium: A) Tetrazolium salt (TTC) may be added to make radiating growth a reddish color B) Special care must be taken to insert the needle straight in and to pull straight out C) The high agar concentration allows limited movement of motile bacteria D) Turbidity is evidence of growth, even if the medium has not turned red

The high agar concentration allows limited movement of motile bacteria

What characteristic of bacterial cell wall determines whether the organism will stain Gram + or Gram -? A) The protein content of the cell wall B) The presence or absence of a capsule made up of carbohydrates C) The thickness of the peptidoglycan layer D) The presence of Mycolic Acid in the cell wall

The thickness of the peptidoglycan layer

Why do acid-fast cells resist decolorization in the presence of Acid-alcohol decolorizer? A) They have a large percentage of protein in their cell wall B) They have a capsule made up of carbohydrates C) They have peptidoglycan in their cell walls D) They produce endospores E) They have mycolic acid in their cell walls

They have mycolic acid in their cell walls

A small drop of water is needed when making a smear from solid media True/ False

True

Complete hemolysis of red blood cells involves lysis with complete degradation of hemoglobin T/F

True

Once your microscope is in Kohler illumination, you should not adjust the field diaphragm, condenser knob, or condenser centering knobs. True/ False

True

When observing a stained bacterial slide under the light microscope, it is necessary to use the 100x objective lens (1000x total magnification) in order to accurately determine the size, staining characteristics and shape of the bacteria True/ False

True

Microbiological growth medium

Tryptic soy broth

Based on the motility test, the bacteria in the left inoculation tube is ___, indicating the presence of ___

motile, flagella