The Ubiquity of Microbes and Their Cultural and Microscopic Characteristics - revised 12/31/08

Ubiquity Experiment Notes:

Work in groups of two

1.) Growing Microbes from Soil or Dust

Using an applicator stick or your fingers if you'd like, place a "pinch" of soil or dust into a tube of sterile tryptic soy broth (TSB).
Replace the cap on the tube and incubate at 35o C (in the incubator).
Please label the tube as we discussed in class - with a small piece of tape onto which you've written your name and section time.

2.) Growing Microbes from Air

Each group of two should take a sterile tryptic soy agar (TSA) plate.
Label the bottom of the plastic plate by writing your names directly on the plastic with your Sharpie pen.
Find an area where you would like to sample the microbial quality of the air. Open the plate and allow it to sit open at your chosen place for at least 20 minutes (be sure to record how long you leave it exposed).
Be creative in deciding which air to sample. For example you can go outside or down to the snackbar or into the restroom.
Several groups may want to cooperate in order to sample several different locations in the same room.
Your instructor will indicate where to place your plates for incubation.

3.)Growing Microbes from your Tabletop

a.) Obtain a TSA plate and divide your plate into 2 equal segments by drawing a line on the bottom. This way you can sample two different environmental surfaces.

b.) Obtain a sterile swab, remove it aseptically from its wrapper and wet it with a drop of tap water.

Take the wet swab and use it to sample some aspect of your workbench.
Now apply the sample to one half of the labeled petri dish. Roll and rub the swab very thoroughly and gently on that segment of the plate so that the whole area of that sector is touched by the swab. (Be sure to label that part of the dish with the name of the sample applied.)
Take another swab, wet it and use it to sample some other area. Again, be imaginative in deciding what you would like to sample.
Apply this second sample to the other half of the petri dish as described above.

4.) Growing Bacteria ON You and IN You

For this experiment you will work individually as follows:

Each person gets one TSA plate. Divide the plate into two equal segments.
Wet a sterile swab with tap water and sample some surface area of your body. Streak this onto one of the segments of your TSA plate.
Repeat the procedure for an internal area of your body except that you probably won't need to wet your swab with water.
Be sure to label your plate appropriately and follow instructions for incubation.

5.) Optional: Activity

You may kiss or lick a TSA or NA plate if you wish.

Interpretation of Results

Can you group the colonies in any specimen according to morphology? Can you come up with some good adjectives to describe colonial morphology?

Attempt to count the colonies and the types of colonies in a specimen.

Are there any pigmented colonies?

Are there any really small colonies?

Be sure to look at any haze on your plates under the magnifying glass or dissecting microscope.

Describe the growth in your test tube. How does your test tube differ now from the way it looked before you inoculated it?

Smell can be an important characteristic! Can you describe the smells you encounter?

Make smears from at least two different colony types and stain them with methylene blue.

Make a smear from your broth. How does this smear differ microscopically from the smears made from isolated colonies?

Can you find any structures that look like endospores in the broth?

Aseptic Transfer from Tube Cultures

The technique used to aseptically transfer microorganisms growing in test tubes is demonstrated on the web. It will also be demonstrated for you in class. This procedure is fundamental and when performed correctly it will help you keep your cultures pure. These techniques will also protect you from contaminating yourself and will keep you free of accidental infection.

Everyone works individually.
Pay close attention to instructor demonstrations and tips.
Learn to hold onto culture tube tops with the hand holding the loop or needle.
Practice the techniques several times with the empty culture tubes provided.
Once you feel like you have it mastered, transfer some Serratia marcescens from a stock culture into two agar slants. Then transfer some Serratia marcescens from a stock culture into a TSB. Incubate the new slant cultures you set up at room temperature (in your tupperware) and the broth culture at 35o C.

Please label tubes with small pieces of tape. Don't write directly on the glass.

Reading Assignments and Questions

Read Tortora assignments listed on syllabus

Why do you think we are doing these experiments?

Name and describe some bacteria, fungi, protozoans that infect humans?

Can you think of any reasons why it might be useful to know the differences between these groups of organisms?

Do you see any representatives of these kingdoms?

What are some of the differences between these types of organisms? Do your drawings from last week and this week show or emphasize any of the differences?

What is meant by "aseptic technique"?

What kinds of things can you do to prevent the contamination of your cultures by organisms on your hands and on your body? What about from organisms on the bench or in the air?

Considering yourself, your activities while at your lab station and the work environment at your lab bench;identify the most likely sources of contamination around you. What kinds of things can you do to minimize the threat of contamination?

What kinds of things can you do to prevent infecting yourself with the bacteria you are working with?

Are these self-protective measures different from the measures you use to protect the purity of your cultures?

How do your precautions and culturing techniques compare with those described in Appendix B of Tortora?

What are Universal Precautions?

What are colonies? How do they arise? What are they made of?

Is a colony synonymous with pure culture?

Will counting the colonies that arise from plating a sample allow you to count the original number of organisms in that sample?

Why do you label the bottoms and not the tops of your petri dishes?

How do you label your tubes?

Lecture Notes

An Overview of Environmental Microbes and the Normal Flora of the Human Body

I. Microbes in the environment

The soil is a vast RESERVOIR of microbes many of which have not even been identified or classified. Fortunately most are not PATHOGENIC to humans. In fact many are vital to the natural processes that shape our environment and others are extremely important in human industry. Examples include:

(1) the NITROGEN FIXING BACTERIA which live in association with LEGUMES and convert N₂ into nitrogenous compounds which are easily absorbed by other plants;

http://academic.reed.edu/biology/Nitrogen/images/part1/bigFL1.jpg

(2) the DECOMPOSERS which return CO₂ back to the atmosphere when they digest organic wastes and dead plants and animals;

The ‘non- decayed’ body of Santa Zita

http://lh6.ggpht.com/_xZW5Pg8r1xU/R_pZNGT_mDI/AAAAAAAACB0/3z_5ucNY8Lw/IMG_2262.JPG

(3) ANTIBIOTIC PRODUCING MICROBES such as Streptomyces sp. and Penicillium sp.

Of course there are a few pathogens which call soil home. These include Clostridium tetani and C. botulinum and this should partly

explain why rusty nails and poorly canned vegetables serve as the source of the diseases caused by these organisms. Pseudomonas aeruginosa is an OPPORTUNISTIC PATHOGEN sometimes responsible for severe infections which are highly resistant to antibiotic therapy. This soil organism is often carried into hospitals with flower arrangments.

Surface water will contain soil organisms but the numbers will vary according to environmental and weather factors. On the other hand, water that moves below the ground usually undergoes a natural filtering process that removes microorganisms. For this reason water from springs or deep wells is often of good drinking quality.

Water is a important source or VEHICLE of human infection if it is contaminated with human or animal waste. Diseases such as typhoid fever (caused by Salmonella typhi or more accurately, Salmonella enterica serovar Typhi), hepatitis, polio, cholera (caused by Vibrio cholera) and viral gastroenteritis (commonly caused by rotavirus and norovirus) are associated with human sewage contamination. In Hawai'i, animal waste contamination of fresh water sources is associated with leptospirosis and giardiasis.

  

The analysis of water pollution will hopefully lead to the discovery and elimination of the pollution source. If the source can be pinpointed to a specific location it is called a POINT SOURCE. Often pollution sources appear to be multiple and spread out over a community. In such instances the pollution is said to come from a NON-POINT SOURCE. For instance, this appears to be a problem in Kailua Bay where run off from the entire community into the various drainage waterways and Kawai Nui Marsh appears to contribute to the high bacterial counts in the bay. This is often a problem all over O‘ahu after heavy rains which results in high bacteria counts in near shore waters.

Some types of water pollution are caused by increasing the level of nutrients in the water. Phosphates in detergents and fertilizers can cause the EUTROPHICATION of lakes and streams leading to dense BLOOMS of algae and cyanobacteria which then cause the death of other organisms in the water. It has been suggested but not proven that fertilizer runoff from suburban yards and golf courses may lead to blooms of Gambierdiscus toxicus, the dinoflagellate algae which is responsible for ciguatera fish poisoning.

II. Microbes of the Human Body

Our bodies have a very rich diversity of microbial flora. It has been estimated that the normal adult has about 1.2 kg of bacteria on and in their body. This would represent about 10¹⁴ bacterial cells of about 400 different species. This NORMAL FLORA is not harmful under normal circumstances and actually performs important functions:

(1) The normal flora serves as a barrier to infection and colonization by pathogenic bacteria;

(2) the intestinal normal flora synthesizes some of the vitamins we require;

(3) the normal flora serves to stimulate the immune system.

The skin is home to a variety of microbial species including Staphylococcus aureus, Staphylococcus epidermidis, Proprionibacterium acnes. These organisms thrive in an environment which is salty and rich in LYSOZYME and lipids which are inhibitory to many other organisms. Of these organisms S. aureus is the most important since it may cause serious skin and systemic infections.

The mouth is home to a large number of streptococci (Streptococcus sp). These organisms make PLAQUE from sucrose as part of their CAPSULE or EXTRACELLULAR POLYMERIC SUBSTANCE. It appears that they do this to help themselves stick to the slippery tooth surface. Unfortunately for us, this plaque acts like a sponge and absorbs the organic acids which these organisms also make when

they FERMENT sugar. The acids dissolve tooth enamel and this leads to tooth decay or DENTAL CARIES. A number of ANAEROBIC BACTERIA are involved in PERIODONTAL DISEASE which eventually can lead to the destruction of roots of the teeth and the gums. The yeast Candida albicans is also commonly present in the mouth in low numbers. The nose is an ideal habitat for Staphylococcus aureus.

The large intestine is home to an enormous number of bacteria whose numbers approach 10¹¹ per gram!!! Of the intestinal flora the COLIFORMS are perhaps the most famous although not necessarily the most numerous. These organisms owe their fame in part to the fact that they are easy to grow and so are very useful as INDICATOR ORGANISMS OF FECAL CONTAMINATION. Technically, coliforms are defined as gram negative rods that FERMENT lactose. The most famous of all the coliforms is Escherichia coli. It is important to know that there are other indicator organisms of fecal contamination such as the fecal streptococci and Clostridium perfringes. These other organisms might be better indicators of fecal contamination in tropical areas like Hawai‘i since there is evidence that some coliforms can survive and multiply in warm, moist fertile tropical soils.

The female genital tract is also colonized by large numbers of normal flora organisms the most important of which are the lactobacilli (Lactobacillus sp). These bacteria are essential for establishing and maintaining the normal acid pH of the vagina and inhibiting colonization by yeast and other organisms. Antibiotics, douches and hormone balance changes which disturb the lactobacillus population often result in vulvovaginal candidiasis caused by the yeast Candida albicans.