Microbiology lab
The _____ should be contacted immediately if you or anyone accidentally consumes or comes in contact with a potentially toxic substance.
National Poison Control Center
What is the purpose of heat fixing? Why did the slides need to be heat fixed during the direct stain but not during the negative stain?
The purpose of the heat fixation in the direct stain was to kill the microorganisms in the smear so they can better adhere to the slide and better uptake the dye. In the negative stain, you would not heat fixate because in negative staining you are not staining the cells themselves but rather you are staining the background. Heat fixing is used when cells are the ones that are being observed/stained. In addition, heat fixing causes shrinkage and/or destruction of the cells which is what we want to avoid in a negative stain.
Basic pH
greater than 7
fixation process
internal and external structures of the cell and the microorganism itself are fixed in place
A basic stain contains ____ charged chromophores.
positively
S. cerevisiae
yeast
Which of the following is NOT equal to 1 meter?
100 millimeter
place the following steps in the correct sequence to prepare the specimen:1- staining2- making smear3-fixing4- air drying
2-4-3-1 making smear air drying fixing staining
As Gram-positive cells age ____, their cell wall ____ causing the peptidoglycan layer to become weak and the crystal violet dye to leak and wash away during the ____step
24-48 hours old; degrades;decolorization
Basic pH
8-14
Bacterial smears are fixed before staining to
A and B.(Kill the bacteria, Affix the cells to the slide)
the best use of a negative staining technique is
A and D (to determine cell size & To determine cell shape)
cells are differentiated after which step in the Gram stain?
Alcohol-acetone wash
Acid spills can be neutralized by adding _____.
Baking soda
Is Crystal Violet a basic substance or an acidic substance? Is Congo red a basic or an acidic substance? Describe how you were able to arrive at this conclusion and relate your findings to your experimental observations.
Crystal violet is a basic stain. Basic stains have positively charged chromophores. The cell membrane is negatively charged thus a basic stain will be attracted to the cell membrane. In this experiment, the cell membranes stained violet meaning that the crystal violet stain is basic. The red Congo stain is acidic. An acidic stain is negatively charged. Since a cell membrane is negatively charged it would repel an acidic stain. In this case, it is evident that the red Congo stain was repelled from the cell membrane thus leaving large red stains around the bacterium.
Is Crystal Violet a basic substance or an acidic substance? Is Congo red a basic or an acidic substance? Describe how you were able to arrive at this conclusion and relate your findings to your experimental observations.
Crystal violet is a basic stain. Congo red is an acidic substance. I was able to come to this conclusion because crystal violet was used in the direct stain. Only basic dyes are used in direct stains, which only color the cell's cytoplasm and leave the background colorless. Congo red was used in the negative stain. Acidic dyes are only used in negative stains.
a spilled base can be made safe by using
Dilute acetic acid
True or false: Graduated cylinders and volumetric flasks are designed to be heated.
False
____ cells and may appear Gram-negative or may appear as both types of cells when stained.
Gram-variable
Why didn't you heat-fix this slide?
Heat-fixing smears can distort cell size and cell shape. Since negative stain slides are not heat-fixed, cell shape and size are not distorted and can be accurately observed. Negative staining is particularly appropriate for visualizing delicate structures like capsules and cells like the spirilla and spirochetes such as Treponema pallidum, the cause of syphilis.
Why is it important to calculate the diameter of the field when first using the microscope?
It is important to calculate the diameter of the field when first using the microscope because you can use it to determine the the approximate size of an object you are examining at the given magnification.
As indicated in the video, what common substance can be used to neutralize a spilled acid?
Keep baking soda on hand to neutralize acid spills.
What morphological characteristics would you use to distinguish between Klebsiella and Bacillus on the slide.
Klebsiella growing under these conditions should have a capsule; Bacillus does not. Klebsiella is also significantly and noticeably shorter than Bacilus.
Why loosen the caps on a tube of agar when heating it?
Loosen the caps of the agar tubes to allow air to escape from the tubes as they are heated. Tilt caps to cover the agar tube while allowing air to escape.
The appearance of gram-negative bacteria after completing the Gram stain
Red
True or false An acidic stain contains negatively charged chromophores
True
The optical microscope is regularly used to identify pathogenic microbes. In 1918 the Spanish Flu infected almost one-third of the world and was thought to be caused by bacteria. However, a virulent virus was the cause of the Spanish Flu. From what we have learned throughout this lab on microscopy, why weren't scientists able to identify the Spanish Flu with an optical microscope?
Unlike many bacteria, the virulent virus that was the cause of the Spanish Flu was too small to be seen by the optical microscope. Many light microscopes allow us to see cells clearly but viruses are very small and therefore unable to be seen by light microscopes including the optical microscope.
True or false:Most pieces of glassware are designed to be heated.
false
bacterial smears are fixed before staining to
kill the bacteria and affix the cells to the slide
Direct stain on bacteria morphology and staining techniques
-Label slide-Draw a circle underneath the slide-Smear the slide with the swab containing teeth and gum swab-Heat fix the sample with the tea candle-Applying crystal violet stain-drying a slide with paper towels after staining.
inserting a glass tubing into a rubber stopper
-lubricate with water and glycerine- use towel- twist the tubing and stopper carefully
Which of the following is NOT correct?
1 nm = 10-6 um
Certain glass objects are not meant to be heated and could shatter if exposed to a heat source. What are two examples of heat-sensitive glassware?
1. Graduated cylinders2. Flasks
List 5 precautions that must be taken before beginning an experiment.
1. Wash hands2. Remove any jewelry3. Tie back long hair4. Wear proper clothing (no loose clothes, close-toed shoes, long pants and sleeves)5. Wear safety googles and gloves
bacterial smears are fixed before staining to
Both Kill the bacteria Affix the cells to the slide
Describe the similarities and differences between the cheek cell wet mount and dental plaque wet mount
Both the cheek cell wet mount and the dental plaque wet mount are best viewed at the 600X magnification. The cheek cells are eukaryotic cells since they are easily shed it's easy to obtain them for observation under the microscope. In the cheek slide smear you can clearly see the nucleus through the microscope. The dental plaque cells are biofilm and are composed of mostly bacteria. Since dental plaque is composed of bacteria it is a prokaryotic cell, meaning unlike the cheek cell the dental plaque is lacking a nucleus and other membrane bound organelles.
The appearance of gram-negative bacteria after addition of the decolorizing agent in the Gram stain
Colorless
Direct stain (+) PH is basic
Direct stain (+) is attracted to the cell membrane (-)
True or false Colorless Gram-negative cells are counterstained in the first step to increase contrast for visualization and identification.
False They are stained with safranin on the final step
Which common and useful staining procedure classifies bacteria into two large groups?
Gram stain
what Gram reaction do you expect from acid-fast bacteria?
Gram-Positive
____ bacteria have an outer cell membrane which is adjacent to and covers the cell wall and is composed of lipids or mostly water
Gram-negative
Cells that are colorless after the decolorization step but counterstain with a second dye (red safranin) are ____
Gram-negative.
Describe the difference between Gram-negative and Gram-positive bacteria. Which type of bacteria are most often treatable with antibiotics and why?
Gram-positive bacteria cells have a thick outer cell wall that is composed of peptidoglycan. Teichoic acids can also only be found in gram-positive bacteria. The size of the gram-positive cell wall is 20-80nm in thickness. Gram-negative bacteria cells have a thin cell wall and only a single layer of peptidoglycan between the inner and outer cell membrane. The outer cell membrane of gram-negative cells contains lipopolysaccharides which are not found in gram-positive cells. The gram-negative cell wall is 2-8nm in thickness. Gram-positive bacteria is often more treatable with antibiotics because gram-positive bacteria has a more superficial cell wall composed of peptidoglycan. It is easier for antibiotics, like penicillin to reach the cell wall of gram-positive bacteria than the gram-negative bacteria where the cell wall is under a strong layer of the plasma membrane.
Cells that retain the primary stain (crystal violet) throughout the Gram stain procedure are ____
Gram-positive cells
acid stain
Laboratory staining process used mainly to identify tuberculosis
acid stain
Negative charge (such as indian ink) will dye the background not the microbes
Negative stain (-) pH is acidic
Negative stain (+) is repelled from the cell membrane (-)
Explain why the cells remain unstained and the background stained
Negative staining is a form of simple staining. The colored component of the stain is negatively charged like the bacterial cell. Therefore, the stain is repelled by the charge of the cell and stains the background, not the cell. Cells will be unstained and appear bright white against a dark back ground
According to the video, why should a used chemical container never be refilled?
Never refill used chemical containers. Used chemicals may be contaminated and you could end up with an unexpected and potentially unpleasant reaction.
Examples of acidic dyes
Nigrosin (black) India ink (black) Congo red (red)
The appearance of gram-negative bacteria after addition of the mordant in the Gram stain
Purple
The appearance of the gram-positive bacteria after adding the counterstain in the Gram stain
Purple
List 5 precautions that must be taken before beginning an experiment.
Read all information before starting your experiments. Get to know your safety protocols. Know the safety protocols for preparation, experiment, and clean-up. Gather, organize and prepare the materials needed for experimentation. Before starting read all info and instructions. Review related MSDS for chemicals used. Complete the emergency contact card. Wear proper clothing such as long-sleeve shirts and pants. Tie and/or move long hair from the face. Remove dangling jewelry. Also, wash your hands. Lastly, wear all safety gear required.
What are small-scale techniques?
Small quantities of chemicals that contribute to the safety of the experiments
What common substance can be used to neutralize a spilled acid?
Sodium bicarbonate or baking soda
What are small-scale techniques?
Techniques used for small quantities of chemicals that contribute to the safety of the experiments. In our HOL kit micro-scale quantities are provided.
What is the major advantage of a differential stain, such as the Gram stain, over a simple stain? Was this observed in your experiment?
The advantage of a differential stain over a simple stain is the differential stain allows you to observe cell morphology and structural components of the cell, while the simple stain only allows you to observe cell shape, size and arrangement. Yes this was observed in my experiment. In experiment one just one stain was used (simple stain, just crystal violet). In experiment two I worked with 4 stains to create a differential stain which allowed me to determine if a cell was gram-negative or gram-positive.
Why should a used chemical container never be refilled?
The container could be contaminated and create an unwanted reaction
What is the purpose of heat fixing? Why did the slides need to be heat fixed during the direct stain but not during the negative stain?
The purpose of heat fixing is to ensure bacteria is kille. It is not necessary to heat fix the slides during the negative stain because the goal of the experiment is to view bacteria that has not been distorted.
Define the three major types of bacterial shapes. Which of these did you observe in the exercise?
The three major bacterial shapes include coccus, bacillus and spirilla. Coccus are spherical or round-shaped bacterium with a diameter of approximately 0.5um. Bacillus are cylindrical or rod shaped bacterium 0.5um to 20um in length. Spirilla are helical or spiral-shaped bacterium approximately 15um in length.
Define the three major types of bacterial shapes. Which of these did you observe in the exercise?
The three most common types of bacterial shapes are bacilli, spiral, and coccus. In this exercise, coccus and bacillis were noted.
Why invert the poured plates while incubating?
To prevent condensation from falling into the microbes, thereby contaminating samples.
True or false Gram-positive bacteria have cell walls that are thick and extensively crosslinked, allowing the dye complex to stay bound and fixed in the decolorization step.
True
True or false: Neutralize acid spills using sodium bicarbonate
True
True or false: A Safety Data Sheet contains information about first-aid procedures, required protective equipment, and health effects.
True
True or false: When conducting experiments that include vigorous exercise, consult a physician first or ask a partner to assist in participating if you are unable to do so.
True
Certain glass objects are not meant to be heated and could shatter if exposed to a heat source. What two examples of heat-sensitive glassware are given in the video?
Two examples used in the video are graduated cylinders and flasks.
Will you need to conduct more than one experiment at a time to meet the assignment due dates? Explain your answer by referencing the completed calendar.
When laying out all the experiments onto my calendar I put them in the weeks based on the syllabus. From there I can see that you do not have to do more than one experiment at a time to get all experiments done by the due date. Based on my calendar, it shows that you are able to get all experiments done before due dates while only doing one at a time. However, while doing Apgar plates you can save yourself time by doing them all together at once and storing them.
Approximately how many total hours should you allocate to complete a lesson that requires an active culture, pouring plates, and incubating microbes?
With a lesson that includes active culture taking approximately 1-3 days, pouring plates taking 1 hour and incubating periods for microbes taking 1-3 days you need to plan for 5 days or 120 hours.
When working with acids, always _____ to avoid chemical splattering.
add acid to water (AA)
bacterial smears are fixed before staining to
affix the cells to the slide
The counterstain in the acid-fast stain is
basic dye
Chemicals in a lab kit may potentially cause _____ if mishandled or consumed.
burns, serious illness, death
In the staining process one of the ions is colored depending on the chemical ____ of the solution.
composition
Examples of simple dyes
crystal violet methylene blue safranin basic fuchsin malachite green.
examples of basic dyes
crystal violet, methylene blue, malachite green, safranin
true or false The color of the chromophores controls how the dye interacts with bacterial cells during the staining process.
false The charge not the color
true or false During staining, a thick film of cells called a smear is applied to a blank microscope slide
false a thin film is applied
A _____ can be used to douse a fire.
fire extinguisher, safety shower, fire blanket, fire blanket and fire extinguisher
process where a flame is used to kill the bacteria and firmly affix the cells to the slide
fixation process
Binary fission
is a method of asexual reproduction during which a single cell (parent cell) will split into two cells (daughter cells). The process then continues as each of these cells divide into two more cells, thereby doubling the population size with each generation. During binary fission cells continuously perform DNA synthesis and divide.
In gram ____ during the decolorization step, the cell membrane is dissolved, exposing the thin cell wall and allowing the crystal violet dye to be washed away
negative
____ stains color only the background or environment outside the cell
negative
Examples of acidic dyes
nigrosin and india ink
acidic
pH less than 7 (pH<7).
The purpose of a mordant in the Gram stain is
prevent the crystal violet from leaving the cells
At the conclusion of experiments involving growing bacterial cultures, _____ should be added to the plates or tubes and everything should be soaked for _____.
pure bleach; 2 hours (pure bleach should be added to the plates or tubes and everything should be soaked for 2 hours)
counterstain in gram staining
safranin
The counterstain in the Gram stain is
safranin. stains all the bacteria. gram-negative appear red.
fixation process
serves to kill organisms, preserve their morphology, and anchors the smear to the slide
Van Leeuwenhoek's microscope was a
simple microscope
Spilled acid can be neutralized with
sodium bicarbonate
True or False Basic dyes which are positive are used for direct stains which only color the cell's cytoplasm and leave the background outside of the bacterial cell colorless
true
True or false You should not hold the slide directly in the flame, too much heat will destroy the sample and potentially break the slide.
true
Ture or False The cell membrane is slightly negative in charge and opposite charges tend to attract, basic dyes thus have a high affinity for the cell's surface
true
true or false Acidic dyes which are negative are used for negative stains in which the dye is repelled from the cell's negatively charged surface
true
true or false heating causes the cell to shrink in size and some cell features may become distorted.
true
