Review of Main Points
You are responsible for everything that we have done, everything that I have covered, and all the reading. Generally speaking, I have pulled the main points out of the reading and put them in
this study guide. I estimate that if you only know this material 100%, you will
get around an 80% on the exam. We also covered many minor points which are
not covered here but are in the online notes. You can count on needing to know the major points for the rest of the term
including lab work & tests. Minor points you may or may not run into again or be tested on (such as laboratory
safety). Here are the main points so far:
- You should know the proper Care and Use of Microscopes.
- You should be able to view slides under the microscope.
- You should be able to find your specimen and draw it under 400x
magnification in 3 minutes.
- Know how to make and why we make Wet Mounts.
- Know how to properly label a Plate, a tube, or a slide.
- Know how to handle a plate and a tube.
- Know how to Streak for isolation of Colonies
- Know some basic sterile techniques used in microbiology
- Know about the Ubiquity of Microbes
- Know what what a general purpose media is and what it is used for.
- Know how to read and interpret colony morphology.
- Know in Colony Morphology: colony shape, margin, elevation (flat or raised or ...), texture (smooth or rough), pigmented or not, translucent or opaque. I expect you to only know intuitive terms & punctiform.
- Know the Three Bacterial Shapes
- Know how to make a Bacterial Smear on a Slide, listing all the steps.
- Know what a simple stain is
- Know that there are two types of simple stains
- Know what is a selective media and what makes it selective
- Know what is a differential media and what makes it differential
- List the selective and differential media that we have used, listing why
it is selective or differential
- encourages some bacteria to grow while inhibiting others
- MacConkey agar (MAC)
- Crystal violet and bile salts generally inhibit Gram positive bacteria
- (Memorization trick, crystal violet stains Gram+ cells, it is
inhibiting Gram+ cell walls in MAC.)
- Phenylethyl alcohol agar (PEA)
- phenylethyl alcohol inhibits the growth of most Gram- organisms
- (Memorization trick, Gram- cells have lots of lipids in their cell
wall, allowing alcohol to readily diffuse thru & inhibit the cell.)
- Mannitol Salt Agar (MSA)
- high sodium chloride concentration inhibits the growth of most
organisms, but not Staph
- the media contains indicators that expose
differences between species.
- MacConkey agar (MAC is both selective and differential)
- Probable coliform -grow and metabolizes lactose (lactose positive)
forming bright pink colonies (probable Gram-).
- Probable noncoliform -grows and is lactose negative, is colorless,
beige or a very dull pink (probable Gram-).
- Mannitol Salt Agar (MSA is selective and differential)
the organism ferments mannitol, an acid is produced turning the
Staph do not ferment mannitol, some pathogenic Staph do.
- Know The Gram Stain
- The Gram stain is the basis for classification and identification in
- It relies on fundamental differences in the cell walls of Gram+ & Gram-
- Make a smear of your bacteria, it must be a light coating of fresh
- Dry and heat fix the slide
- To Stain with the Gram Stain (which is a differential test or differential
- stain w/ crystal violet for 1 min
- wash slide w/ water
- fix crystal violet by adding Grams iodine for 1 min (forms a complex w/
- wash with water
- decolorize by washing w/ ethanol or ethanol-acid or ethanol-acetone
mixture (we used ethanol-acid)
- wash with water
- counter stain with safranin for 1-2 min
- wash with water
Things that can go wrong with a Gram stain
- If too many cells, it will stain gram positive, purple, even when it
really is not because not all the crystal violet will wash out when it
- If bacteria are old, it may not stain properly from dead & sick cells.
- If the decolorizing step (alcohol wash step) is too long or strong, it
will wash out the crystal violet, even when it should not.
- If the decolorizing step is too little, not all of the crystal
violet-iodine will wash out when it should.
- Some bacteria simply do not stain well (consistently) w/ the Gram stain.
|Appearance of the Gram positive coccus
and Gram negative bacillus at different stages of the gram staining
procedure are illustrated on the right.
- Gram+ cell membranes retain the crystal violet-iodine complex, so
they stain purple (the safranin is masked as it is lighter in color)
- Gram- cells loose the crystal violet-iodine complex in the
decolorizing step, so they stain pink/red from safranin.
- Why do the above occur?
- Basically it is staining cells w/ two stains to look
depends on the purpose of the stain
- We will cover two differential stains:
- the Gram
to divide cells into Gram- or Gram+)
- the Endospore Stain (Purpose:
to stains cells differently w/ or w/o endospores). We will cover
this stain later in the term.
You should know how to use a pipette.
Know how to read and use a pipette correctly.
Know how to get the last drop out.
Know how to use a pipette aseptically.
Serial Dilutions (weeks 5-7)
Know the purpose of serial dilutions.
If given the dilution and the bacteria counts on a
plate, be able to calculate the concentration of bacteria. You can
assume that you will always be plating 1 ml and using dilutions based on 10
to make the math easier.
Motility in bacteria (wk5 & 7 or 8)
Can be determined from the Motility Agar Test, Exercises 120, Atlas 67
uses a very thick liquid and a stab of bacteria.
If the bacteria are not motile, they will only grow
around the stab. If they are motile, they will spread and grow away
from the stab
How else can we determine if our bacteria are motile?
Carbohydrate Fermentation Test or Phenol Red (PR) Broth
- Is a fermentation media that includes a series of tubes each with a
different sugar and with the pH indicator Phenol Red.
- You inoculate bacteria into each tube, if it ferments that sugar, an acid
will build up, changing the color of Phenol Red. Hydrogen and carbon
dioxide gas and alcohol may also be produced by fermentation.
- Test is used in identifying Gram- bacilli, especially Enterobacteria
- Results can be positive (or negative) for acid production, gas production,
(and alkaline product production though this is seldom utilized).
Results are from the tube color and presence of bubbles.
Growth Patterns in Solid And Liquid Culture
- is an undefined and differential media that is used to help differentiate
and identify Enterobacteria, Clostridium, and Lactic acid bacteria.
- A number of results may be obtained in the Litmus Milk test, but there
are four main reactions: lactose fermentation, litmus reduction, casein
coagulation, and protein hydrolysis. Some or all of these reactions
may occur at the same time, and many of them have multiple or similar
- Know how to read the test if provided with a tube and a table.
A number of characteristics may be observed when growing bacteria in both
solid and liquid media. One may ascertain the oxygen requirements of an
organism, the motility of an organism, the appearance of the growth pattern,
and in some cases to some extent the chemical makeup of the cell wall.
Although useful, obtaining these characteristics in this way is not always
100% reliable, at least not when trying to identify an organism. Yet,
they are useful and cheap tests for confirmation purposes of another test.
The Deep Stab (Growth Patterns in Solid Culture).
Growth in Broth (liquid) Culture.
The Deep Stab is a tube filled with a medium containing agar. It is
inoculated with a needle to observe the growth patterns in the agar.
The presence and location of growth in the tube then indicates the oxygen
requirements for the organism.
- A Broth Culture is a tube filled with a liquid medium. It is
inoculated to observe the growth patterns in a liquid medium.
- One may determine the location of growth, the appearance of growth,
and whether their are sediments or floating material. One may even be
able to determine the motility with a fair degree of accuracy by looking
at the growth appearance. Motile organisms may have growth throughout
the tube, whereas non-motile organisms will have growth in one location,
usually sedimented on the bottom.
Blood Agar Plate (BAP)
TSI (Triple Sugar Iron) and H2S Production
- also used to grow fastidious organisms requiring a rich media (blood)
- It is differential in allowing the detection of different
types of hemolysis (rupturing the blood cells)
clear zone -RBC hemolyzed completely (Beta-hemolysis,
green zone -RBC partially hemolyzed (Alpha-hemolysis)
no change or zone -RBC are not hemolyzed (Gamma-hemolysis or no
- Purpose -to identify & differentiate members of Enterobacteria (enterics)
from other Gram- bacilli.
- TSI is a rich medium containing a pH indicator, peptones, several
sugars (testing for fermentation) and sulfur (tests for sulfur
reduction). The slant is to allow for aerobic growth, the butt to
allow anaerobic growth.
- Many combinations of results are possible, and this is a complex test.
- Be able to read the test if given a tube and the table