Key to Past Lab Test 1

Gram stain — Name the mordant.

odine

Gram stain — What results would be wrong if the primary and secondary dyes were switched?

Gram + would be correct (purple) as both dyes adhere.

Gram - would be wrong (purple) as they would lose the primary (pink) dye and pick up the purple dye used last.

Acid-fast stain — Name the primary dye.

Carbol fuchsin

Acid-fast stain — What results would be wrong if the decolorizer from the Gram stain was used?

All gram + cells, not just the acid fast ones, would keep the primary dye and be pink if this weaker alcohol was used.

Spore stain — Name the decolorizer.

water

Spore stain — What results would be wrong if the decolorizer from the acid-fast stain was used?

No changes from normal would be seen, spore coats are thick and impermeable, any dye inside would stay.

Capsule stain — What is the charge of the secondary dye?

positive

Capsule stain — What results would be wrong if the slide was rinsed between reagents?

Since no heat fixing occurs, cells would be washed away along with dyes during rinsing; nothing on slide upon viewing

Spore stain — Name the secondary dye.

safranin

Spore stain — What results would be wrong if steaming was done during secondary dye application?

The pink dye would enter the endospore along with the green dye already there, so the spore would be a combined brown-ish color with a pale pink vegetative cell.

Negative stain — What is the charge of the primary dye?

negative

Negative stain — What results would be wrong if a heat-fixed smear was used?

The cell size and shape would be distorted by melting the bacteria onto the class.

If you grabbed a dye without looking to see what it was, and after performing a simple stain on your heat-fixed smear, no color is seen on the slide. What is probably true of the dye you used:

You grabbed an acidic (negatively charged dye) which was repelled by the negative charge of the bacterial cell surface, and thus did not color the cells.

You did the spore stain procedure on a mixture of acid fast and non-acid fast cells. What would the color of each type of cell be?

Acid fast cells should be green (heat forced primary dye malachite green inside).

Non-acid fast cells should be pale pink (water easily removes weak green dye, and they take up safranin)

Your unknown is described as having a sub-terminal, small, round endospore. Draw what that

would look like:

You see a mixture of different cell types under the microscope when you prepared a simple stain from the culture tube at your station in lab. You are the last lab of the day to meet. Why do you have a mixture of cells instead of only one type? (Hint: what did someone do wrong, WHEN?)

Someone contaminated the culture in an earlier lab, and the contaminant reproduced to be present in large numbers by the end of the day. They may have failed to flame their loop before entering culture, or left the tube open while making the smears.

You did an acid-fast stain on your unknown. Instead of bacterial cells, you see a bunch of trash under the microscope. What may have been done wrong to cause this?

You probably heat-fixed the TOP of your slide and burned the bacteria to ashes. (or you did not wash the slide, or you steamed primary dye too long/without paper slip on top)

You do your gram stain meticulously, but after drying and viewing, there is obviously NOTHING on the slide. What did you probably forget to do?

You forgot to heat-fix the smear before staining.

Which two stain procedures require heat during the primary stain application? Why is this step necessary for each stain?

Stain Procedure 1:
Acid-fast stain

Reason:
Heat melts the mycolic acid (waxy layer) in the cell wall, allowing the primary dye to penetrate under the wax layer. Once inside, the dye is trapped and cannot be removed, even by the harsh acid-alcohol decolorizer.

Stain Procedure 2:
Spore stain

Reason:
Heat loosens the multiple layers of peptidoglycan and protein in the spore coat, allowing the primary dye to enter. When the layers re-harden, the dye becomes trapped inside the spore.

Glass slides are heated at two different times during bacterial smear preparation, for different purposes and using different methods.

Heating procedure 1 is done when and how? Reasons?

Heating procedure 2 is done when and how? Reasons?

1. When and how? Meticulously cleaning slides: Flame 2X front and back

Reasons: Remove oil and link so that smear will be able to spread and extra trash won’t be seen.

When and how? Heat-fixing is done after smear air dries: 2X on back only

Reasons:

1-Stick bacteria onto slide

2-Kill bacteria

Which of the areas below would you hypothesize to possess the MOST bacterial cells, when swabbed and inoculated onto culture media? Give at least 3 reasons to explain WHY this location is hospitable to bacteria.

• Cat’s water dish (plastic)

• Cell phone screen

• Doorknob to lab room

• Cat’s water dish (plastic)

It is 1-moist, 2-has bacteria introduced from cat’s mouth frequently, 3-warm (room temp), 4-organic material (falls in from cat’s mouth, open to air), 5-pH is probably neutral from water pH, 6-No metals or antibiotics/disinfectants included, 7-hypotonic to bacterial cells so cell wall protects from overexpanding and dying.

True or False: After focusing on a specimen with the high-dry objective, before switching to the oil immersion objective, you should always lower the stage.

False;

Leave the stage at the correct level established on the earlier objective.

True or False: When performing a bacterial smear for staining, the culture tube should be flamed and re-capped after smearing the specimen on the slide.

False;

Before

True or False: At the end of the capsule stain procedure, the capsule will be colored by the 1o

False;

Uncolored

Applying the secondary dye correctly is the most important step to ensure accuracy of the Gram stain.

False;

Decolorizing agent

You should only need to adjust the iris diaphragm aperture once, on low power.

False;

For each different objective you put into place

Washing hands with bar soap and a scrub brush will increase the total number of bacteria on the surface of the hands.

True

The waxy component of the cell wall found in acid-fast bacteria only is teichoic acid.

False;

Mycolic acid

The inoculating loop is used to obtain specimen from the tube when making an aseptic smear from slant cultures.

False;

Needle

The light intensity should be at its highest when you are using the highest

power objective on the microscope.

True

The drop of immersion oil applied to the slide greatly increases both magnification AND resolution of the specimen being viewed.

False;

Resolution only

Washing the hands with liquid soap and water is more effective at reducing the number of extraneous organisms on the surface of the hands than waterless hand sanitizer.

True

The cap of a test tube should be placed gently on the counter when removed for obtaining a specimen from the culture for preparing a stain.

False;

Held in the pinky finger of your dominant hand

When making a negative stain, the dye remains on the background of the slide due to its attraction to the charge of the glass itself.

False;

The fact that you do not rinse the slide

Name AND give the function of the microscope part at the pointer

Iris diaphragm, opens and closes to narrow the wide beam of light on the specimens on the slide for each magnification. (Improves resolution by cutting out light from edges of image.)

Give the total magnification in use, showing calculations

40X (objective) multiplied by 10X (ocular) gives a total of 400X magnification

When would you use this item? What is its purpose?

(bibulous paper pad) Used to dry slides once staining procedures are finished.

What is the name of this item? From what type of culture medium is it used to obtain a specimen?

Inoculating loop; liquid or broth cultures (also water on slide for slant cultures when making smear)

Record the microbiological term for the shape of this organism. Is the dye used here basic or acidic?

Micrococcus negative slide seen) Cocci; acidic

Give the gram reaction, morphology, and arrangement of the organism shown.

(B. subtilis) gram + bacilli, singles or short chains

Give the gram reaction, morphology, and arrangement of the organism shown.

(Neisseria) gram – cocci, diplo

If you performed a gram stain on the cells which appear pink with the acid-fast stain here, what gram reaction would it have? What do you know about its cell wall that leads you to this decision?

(acid fast cells) gram +, the wall is thick peptidoglycan with mycolic acid embedded within it, NOT thin peptidoglycan with an LPS layer

If this organism was isolated from the sputum of a patient who was coughing up rust-colored sputum, what genus name and disease is this likely to be?

(same slide) Mycobacterium, tuberculosis

Record the three specific characteristic features of this particular organism's spores

(B. sphaericus spore stain) Large, oval, terminal spores

Which of the smears shown here would be the best to use for gram staining? Why?

Smear C is perfect, thick enough to find, but not so thick that cells are on top of each other, preventing viewing of individual cells and preventing appropriate decolorization.

Which side of the petri dish seen here would be likely to have more extraneous organisms, and which side would have more normal flora? Briefly explain why

The “Unwashed” side would have normal flora (always present) as well as extraneous organisms you have picked up recently from touching things.

The “Washed” side would have only normal flora (extraneous on surface, washed away down the drain)