Microbiology Lab Exam 2 (Case Study Exercise 8)
Palisades
Cell ends attach side to side, like a zig zag
What are the steps to a smear preparation from liquid media?
Flame inoculating loop and allow to cool Obtain two loopfuls of bacteria from culture and place in the center of a clean slide Spread culture (quarter-size) Allow to air dry
What is the most important differential stain in Microbiology?
Gram stain
What are staining techniques in the laboratory designed to do?
Increase contrast between bacterial cells and their backgrounds
What is responsible for coloring of the cell?
Ionic interactions between chromospheres and bacterial cells
What is the function of the counterstain?
Used to stain the now colorless Gram (-) cells pink Gram (+) cells are stained pink at this point too but since purple is dark than pink, the color does not show
What is simple staining?
AKA positive stains that will be attracted to the bacterial cell, staining it and leaving a clear background
What is negative staining?
Bacteria are negatively charged Negative stains will only color the background because the cell will repel the anions in the stains
What are some examples of positively charged or basic stains?
Crystal violet, Methylene blue, Malachite green, and Safranin
What are the steps to a smear preparation form a solid media?
Flame inoculating loop and allow to cool Obtain two loopfuls of water and place in the center of a clean side Flame inoculating needle and allow to cool Obtain small amount of solid culture and mix it into the two loopfuls of water on the center of the slide (should appear cloudy) Spread culture (quarter-size) Allow to air dry
What is the function of the mordant?
For use after the primary stain Binds with the crystal violet to chemically alter the shape of the dye's molecules so the primary stain will be stuck onto the cells of the organism
S. aureus
Gram (+) Irregular clusters (grapes)
L. innocua
Gram (+) Singular rod
B. megaterium
Gram (+) Streptobacillus
M. luteus
Gram (+) Tetrad
E. coli
Gram (-) Singular rod
S. marcescens
Gram (-) Singular rod
What is done for staining stains that both kills the bacteria and sets them to the slide?
Heat fixation
How is cellular arrangement determined?
How they divide and how they stay attached after division determine their arrangement
What is meant when a bacteria is pleomorphic?
Its shape varies even in the same sample
Which stains can be used for negative staining, and why can these same stains not be used for simple staining?
Nigrosin and India ink Can't be used for simple stains because simple stains are used to stain the bacterial cells themselves Nigrosin and India are negatively charged stains and they repel the negatively charged bacteria and cannot stain the bacterial cells themselves
What color are Gram (+) cells?
Purple -> thick peptidoglycan layer
What is the difference between a simple stain and a differential stain?
Simple stains only require the use of one stain whereas differential stains require a primary stain and a couterstain
What does it mean when a bacterium is classified as a bacillus?
Singular, rod-shaped
What does it mean when a bacterium is classified as a cocci?
Singular, spherical (rounded)
What are the steps to negative staining bacteria?
A small drop of Nigrosin or Indi ink is place near the end of a clean slide Using an inoculating needle, add a small amount of B. megaterium to the ink and swirl in the dye Use a second clean slide and spread the dye, backing up into the stain and then sliding forward so as to thin out stain Allow stain to air dry and then examine under low/high/oil
What is the function of the primary stain in a Gram stain?
Both the Gram (+) and Gram (-) cells are stained at this point
How do Gram stains work?
Cells are first stained with Crystal Violet (*primary stain*) -> both Gram (+) and Gram (-) are purple at this point Rinsed with water for 2 seconds Gram's iodine is applied as a *mordant* which serves to essentially set the stain onto the cell -> iodine bind with the Crystal violet to create an insoluble complex within the thick peptidoglycan layer of Gram (+) cells Cells are then *decolorized* with ethyl alcohol -> alcohol dissolves the lipids found in the outer membrane of Gram (-) cells, allowing crystal violet to escape and decolorize; Gram (+) remains purple A *counterstain*, Safranin, is apply to increase the contrast of the now colorless Gram (-) cells making them pink -> Gram (+) are dyed pink as well but since purple is dark than pink, you can't see the pink
Which shape of bacteria produces more arrangements?
Cocci
Based strictly on cellular morphology, which bacterial species used in this exercise is most easily differentiated from the others? Why?
Cocci and bacilli can most easily be differentiated from each other S. aureus differs from E. coli in that S. aureus is irregularly clustered in cocci and E. coli is rod-shaped S. aureus is Gram (+) whereas E. coli is Gram (-)
What are bacteria that are intermediates of cocci and bacillus classified as?
Coccobacilli
What is chromosphere?
Colored portion of a stain molecule that carries an electrical charge
What is the most critical step in a Gram stain?
Decolorizing step -> can either take away the crystal violet from Gram (+) and Safranin will stain them pink and render a false Gram (-) or done too little and the Gram (-) stain won't even be seen rendering a false Gram (+)
What are the three arrangements seen in bacilli?
Diplobacilli Streptobacilli Palisades
What is the arrangement called when a coccus divides along a single plane?
Diplococci
What is the function of the decolorizer?
Dissolves the lipid outer membrane in Gram (-) so the purple primary stain will be stripped from from Gram (-) cells Gram (+) cells do not have an outer membrane which is why those cells remain stained with purple
What the purpose of increasing contrast between cell and background?
Easier to determine size, shape, and arrangement of a particular bacterium Negative stains gives a more accurate determination of size
What is the arrangement called when it divides in several planes?
Irregular clusters called staphylococcal
What are the steps to Gram stains?
Label four slides Prepare heat fixed smears for each organism to be examined -> 2 loopfuls of liquid culture to center, spread, and air dry or 2 loopfuls of water and 1 needle of bacteria, mix, spread, and air dry Allow the smears to air-dry completely before running the slides three times under Bunsen burner Flood the slide with Crystal violet (primary stain) for 30 sec Briefly use water to wash excess stain from slide (3-4 sec, maybe even 2) Flood the slide with Gram's iodine for 1 min Decolorize smear with ethyl alcohol for 8-12 sec Immediately rinse with water to stop decolorizing effect of the alcohol Flood smear with Safranin for 1 min Blot slide with bibulous paper to remove excess water Examine under low/high/oil
Streptobacilli
Multiple bacilli that dd not separate after division
What two staining techniques are appropriate for determining the shape and arrangement of a bacterial species?
Negative stain -> don't require heat so cell shapes are shrunken Differential stain -> Gram stain is most widely use
Why are negative stains more useful in determining size than simple stains?
Negative stains don't require heat fixation When the stains are heat fixed, they shrink Simple stains require heat fixation and shrink the cells whereas negative stains don't need heat fixation
What are two examples of negatively charged or acidic stains?
Nigrosin and India ink
What color represents Gram (-)?
Pink
What color are Gram (-) cells?
Pink -> thin peptidoglycan layer and lipid outer membrane
What are the steps to simple staining?
Prepare a smear of L. innocua by adding two lapfuls of culture to center of a clean slide (flame loop each time you enter culture tube) Spread culture (quarter-size) in the center of the slide Allow smear to air-dry completely (5-10 min) Once dry, heat fix by passing the slide through Bunsen burner three times Flood smear with crystal violet for 1 min Gently water off stain from smear (3-4 sec) Blot slide with bibulous paper to remove excess water Examine under low/high/oil
What color represents Gram (+)?
Purple
What is meant by the term morphology?
Refers to the shape of a bacterial cell
What does the "Gram reaction" of a cell refer to?
Refers to what colors the bacterial cells stain and how thick their peptidoglycan layer is
What is the arrangement called when it results in a cuboidal packet if 8 cells?
Sarcina
What does it mean when a bacterium is classified as a spirilla?
Singular, spiral-shaped
What are flexible spiral bacteria classified as?
Spirochetes
What is the arrangement called when the cells continue to divide without separating?
Streptococci
What is the arrangement called when a coccus divides in two planes?
Tetrad
How are differential stains useful?
They provide information regarding size, shape, arrangement Also because they highlight difference between each organism, they can show structural differences that exists between them
What is the purpose of rinsing off the decolorizer with water?
To stop the effects of the decolorizer so you don't decolorize too much and end up stripped the smear of all stain
How do rod-shaped bacteria divide?
Transversely -> in half down the center
Diplobacilli
Two bacilli that did not separate after division
What are short, curved rod-shaped bacteria classified as?
Vibrios
What are three important factors regarding Gram stains?
Young cultures should be used instead of older cultures as they tend to have changes in their peptidoglycan layers as they age Smears should not be too thick as they can entrap crystal violet so it can't be removed by the alcohol causing false Gram (+) results Decolorization must be done for an appropriate amount of time because if not done long enough, Gram (-) cells will be left purple causing a false Gram (+) results and if done for too long, crystal violet will be stripped from the Gram (+) cells and they will end up staining pink causing a false gram (-) result