Bacterial Cell Shapes and Arrangements

When stained, most bacteria will be observed to have one of three shapes:

• Coccus (spherical)

  

• Bacillus (rod-shaped)

  

• Spirilla (spiral)

  

Cocci

Examples of diplococci include Streptococcus pneumoniae, which is Gram positive, and

Neisseria gonorrhea, which is Gram negative.

Some cocci occur in chains. Examples of these include Streptococcus pyogenes, which is Gram positive.

Staphylococcus aureus, are Gram positive cocci arranged in clusters.

Bacilli

Escherichia coli, left, is a Gram negative rod.

Bacillus subtilis, right, is a Gram positive rod. 'Bacillus' in this case is the bacterial genus name and is capitalized and italicized.

Members of the genus Vibrio are Gram negative bacteria whose shapes are categorized as rods. However, they are distinctly comma-shaped.

Spirilla

• Spirilla have a spiral or helical shape. They resemble corkscrews.
• They often have an axial filament.
• They are difficult to stain and best viewed under the phase contrast microscope.

 

Borrelia burgdorferi is observed using the phase contrast microscope, above.

• 'Dichotomous' means having two categories or classifications.
• A dichotomous key is based on successive questions.
• In microbiology, it is used to identify an organism.
• Each question has two possible answers.
• Each of the two possible answers directs the individual in a completely different direction to different questions
• Eventually, no more questions can be asked, and the identity of the organism is revealed.
• In microbiology, the first question to be asked is always 'what is the Gram stain reaction?'
• If the answer is 'Gram positive,' then the individual will follow the series of questions that are asked of all organisms staining Gram positive.
• If the answer is 'Gram negative,' then the individual will follow the series of questions that are asked of all organisms staining Gram negative.

Instructions for Gram Stain:

Materials:

Inoculating loop

Glass slide

Slide holder

Glass marking pen

Bunsen burner

Flint lighter

Bibulous paper

1. Label your slide, if you plan on keeping it.

2. Place a small drop of .85% saline on the slide.

3. Sterilize your inoculating loop and select a colony. Pick it up and mix it with the saline.

4. Allow the saline/colony solution to dry fully.

5. Heat fix it.

6. Gram stain it:

• a. Crystal violet, one minute. Rinse with water.

• b. Iodine, one minute. Rinse with water.

• c. Alcohol, until runoff is clear OR 20 seconds, whichever is shorter. Rinse with water.

• d. Saffranin, one minute. Rinse with water.

7. Blot dry, using bibulous paper.

8. Coarse focus on 10x; fine focus, using oil, on 100 x.

Serial Dilution Demonstration

Sometimes it becomes necessary to perform a colony count. A colony count is an actual count of the total number of colonies of bacteria in a certain volume of specimen. Volume is measured in milliliters. Because bacteria can be present in very large numbers and counting these large numbers could lead to errors, serial dilutions of the original specimen are set up for culturing, which make counting more reliable. Dilutions are expressed as the ratio of the quantity of the solute (the bacterial specimen) contained in the solution (the sterile diluent plus the solute). To perform a serial dilution for a colony count,

• Accurately measured volumes of sample are mixed with a large volume of sterile diluent.
• In this lab, one mL of the original specimen is placed into 9 mL of sterile nutrient broth. This now becomes a 1:10 dilution. This means there is one part solute (bacterial sample) in ten parts of the solution (the solute PLUS the solvent).
• This can be expressed in a variety of ways:
• 1:10 dilution
• 1/10 dilution
• 10^-1 dilution
• 1 x 10^-1 dilution
• All of the above expressions mean the same thing: there is one part of the bacterial sample in ten parts of the solution.
• Now, we take one mL of the 1 x 10^-1 dilution sample and add that to another test tube containing 9 mL of sterile nutrient broth. This now becomes a 1 x 10^-1 dilution times the 1 x 10^-1 dilution from before. Attached to this lab is a copy of how to multiply using scientific notation. You may want to refer to it. To determine the final dilution of this sample, multiply 1 x 10^-1 by 1 x 10^-1 to get 1 x 10^-2.
• 1 x 10^-2 is the same as 10^-2, which is the same as 1:100, which is the same as 1/100, which is the same as .01 dilution.
• If we take one mL of the 1 x 10^-2 dilution and add that to another test tube containing 9 mL of sterile nutrient broth, we have made a 1 x 10^-1 dilution of the 1 x 10^-2 dilution. This means the dilution of this solution is equal to 1 x 10^-3.
• We can express 1 x 10^-3 as 10^-3, 1:1000, 1/1000, or .001. It is all the same.
• Now, taking one mL of the 1 x 10^-3 dilution and adding it to another test tube containing 9 mL of sterile nutrient broth, results in a solution that has been diluted 1 x 10^-1 by a 1 x 10^-3 dilution. The final dilution of the solution in this test tube is equal to 1 x 10^-4.
• 1 x 10^-4 is the same as 10^-4, which is the same as 1:10,000, which is the same as 1/10,000, which is the same as .0001.
• Refer to the diagram below for clarification.

The original sample is black, in the test tube labeled "1.0." This represents the undiluted specimen. Each subsequent test tube contains 1/10 of the amount of the previous test tube.

What would the dilution amount of the sixth test tube be equal to?

A Review of Scientific Notation

Precision in Communication

Within the laboratory, it is customary to write numbers using scientific notation, also known as exponential notation. There are many advantages to using this system, especially as the field of microbiology deals with very small numbers.

Numbers written in scientific notation take the form of M x 10ⁿ.

• M represents a number between 1 and 10 and includes all the significant figures that are known about the number.
• The exponent, n, locates the decimal point and is obtained by counting the number of places between the original decimal point of the number and the new decimal point that is present in the scientific notation format.
• When writing the value for n, the location of the decimal point is represented by a positive sign if the number is greater than 1, and a negative sign if the number is less than one. When the number is equal to one, it is written as 1 x 10⁰.
• For example, 1000 is written as 1 x 10³; .001 is written as 1 x 10^-3.

To express numbers in scientific notation, remember that every number consists of the product of:

• a number between 1 and 10 containing the proper number of significant figures, and
• a power of 10, indicating the location and number of decimal places.

One of the advantages to using scientific notation is that addition, subtraction, multiplication, and division of very cumbersome numbers become comparatively simple.

• All exponents must be the same before the values of M can be summed or subtracted.
• Example: 6.5 x 10² + 2.0 x 10⁰ - 3.5 x 10^-1 =
• Change all numbers such that the exponents are the same.
• One way to do this is 6.5 x 10² + .02 x 10² - .0035 x 10² = 6.5165 x 10²
• Another way to do this is 650 x 10⁰ + 2.0 x 10⁰ - .35 x 10⁰ = 651.65 x 10⁰
• To put this number back into standard scientific notation, simply re-write it as 6.5165 x 10².

• In multiplication, exponents of like bases are added (we use base 10 for M); in division, exponents of like bases are subtracted. The values for M are multiplied or divided, as indicated.
• Multiplication example: (3 x 10⁵) x (2 x 10⁴) = 6 x 10⁹
• To square, square the number M and multiply the exponent by 2; then continue:
• (3 x 10⁵) x (3 x 10⁴)²= (3 x 10⁵) x (9 x 10⁸) = 27 x 10¹³
• Division example: (6 x 10⁴)/(3 x 10²) = 2 x 10²

Take Out Food for the Brain:

You are what you eat, and this apparently holds true for cattle, where a recent study has demonstrated that enterohemorrhagic Escherichia coli in a cow's gastrointestinal tract can be virtually eliminated by a simple change in the cattle's diet.

E. coli inhabit the intestines of cattle and other animals. Certain strains of E. coli found in cattle contain genetic information that codes for the production of a toxin, called Shiga-like toxin. This toxin is a protein that damages the cells and blood vessels that line the walls of the human intestine, leading to a hemorrhagic enterocolitis, which can be deadly. The most common enterohemorrhagic E. coli in the U.S. are called E. coli O157:H7, and they are considered very dangerous. E. coli O157:H7 is harmless to cattle, as are the other E. coli variants.

E. coli, along with other kinds of bacteria are also normal flora in the human intestine. E. coli are humans' main source of Vitamin K and B-complex vitamins, and their presence is necessary for proper growth and development. Serotyping, toxin detection, and other laboratory tests are used to distinguish between pathogenic and non-pathogenic E. coli.

A team from Cornell University and the U.S. Department of Agriculture reports that cattle fed mostly grain to fatten them up for sale are more likely to develop pathogenic strains of E. coli than cattle fed hay and grass. Even switching the cattle to hay and grass a few weeks before slaughter will almost eliminate the presence of pathogenic strains.

This discovery could reverse the increasing incidence of meat contamination by enterohemorrhagic E. coli. However, regardless of whether cattle ranchers change their practices, the public can protect itself from pathogenic E. coli and other food-borne pathogens by washing all utensils and meat preparation areas carefully and cooking ground meats until the juices run clear. And always, always, always wash hands well before and after handling meat!

How is it possible that E. coli are necessary to humans' welfare and at the same time can be deadly to humans? You tell me!

"The whole world is our dining room, but be careful: it is also our garbage can."

: Brilliant Thoughts--