1. “What causes Yogurtness”?
Microbes and Health:
“What causes Yogurtness”?

2. Bacteria, Bacteria, Bacteria
The single most successful life form on earth
Prokaryotic organisms
Exist in soil, water, in and on animals, plants and humans
Can orginize as
single units, pairs, long strings, helical shapes, twisted spirochetes
Divide by binary fission (some every 20 min!)
Colonies originate from one bacterial cell (clonal growth) and can have different shapes
Gram’s stain dye is taken up by bacteria with thick cell walls (Gram + or -)
Keep slide in ppt but do not plan to present

3. Bacteria Classification
Three types based upon their shapes…
Bacteria Classification
Coccus
Bacillus
Spirillum
Spherical shape
Oval or rod shaped
Spiral shaped
“strepto” from Greek streptos meaning ‘twisted chain’
“staphylo” from Greek staphule meaning ‘bunch of grapes’

4. Good Bacteria, Bad Bacteria Bacteria as Pathogens
Cholera – Vibrio cholerae
Typhoid fever – Salomonella typhi
Anthrax – Bacillus anthracis
Tuberculosis – Mycobacterium tuberculosis
Beneficial Bacteria
Rhizobia – soil bateria important for nitrogen fixation
Human bacterial flora – ,00 species of
bacteria live in the human body
Lactobacillus species – convert milk to lactic acid
Digestion of oil spills -
Marine bacteria: Acinetobacter calcoaceticus RAG-1
Genetic engineering – use of E.coli in industry and
reasearch
Rhizobia (from the Greek words rhiza = root and bios = Life) are soil bacteria that fix nitrogen (diazotrophy) after becoming established inside root nodules of legumes (Fabaceae). The rhizobia cannot independently fix nitrogen, and require a plant host. Morphologically they are generally gram negative, motile, non-sporulating rods.
Acinetobacter calcoaceticus RAG-1 is a marine bacteria which can utilize the hydrocarbons in oil as a source of carbon. When these bacteria are grown in a carbon minimal medium, they will breakdown hydrocarbons. RAG-1 releases an emulsan - a polysaccharide that will emulsify oil. Emulsan accumulates on RAG 1 cell surfaces as minicapsules and is released into the media as an active emulsifier as the cell growth approaches the stationary phase. The absence of a carbon nutrient source accelerates the release of emulsans.
Salmonella typhimurium

5. Koch’s Postulates

6. Robert Koch Robert Koch (pronounced “coke”)
- German physician and bacteriologist
- Lived
Developed a criteria for determining whether a given bacteria is the cause of a given disease:
Known as Koch’s Postulates
In 1890 the German physician and bacteriologist Robert Koch set out his celebrated criteria for judging whether a given bacteria is the cause of a given disease. Koch's criteria brought some much-needed scientific clarity to what was then a very confused field.
Koch's postulates are as follows:
The bacteria must be present in every case of the disease.
The bacteria must be isolated from the host with the disease and grown in pure culture.
The specific disease must be reproduced when a pure culture of the bacteria is inoculated into a healthy susceptible host.
The bacteria must be recoverable from the experimentally infected host.
However, Koch's postulates have their limitations and so may not always be the last word. They may not hold if:
The particular bacteria (such as the one that causes leprosy) cannot be "grown in pure culture" in the laboratory.
There is no animal model of infection with that particular bacteria.
A harmless bacteria may cause disease if:
It has acquired extra virulence factors making it pathogenic.
It gains access to deep tissues via trauma, surgery, an IV line, etc.
It infects an immunocompromised patient.
Not all people infected by a bacteria may develop disease-subclinical infection is usually more common than clinically obvious infection.
Despite such limitations, Koch's postulates are still a useful benchmark in judging whether there is a cause-and-effect relationship between a bacteria (or any other type of microorganism) and a clinical disease.

7. Koch’s Postulates
The microorganism must be found in all organisms suffering from the disease, but not in healthy organisms.
2. The microorganism must be isolated from a diseased organism and grown in pure culture.
3. The cultured microorganism should cause disease when introduced into a healthy organism.
4. The microorganism must be again isolated from the inoculated, diseased experimental host and identified as identical to the original specific causative agent.

8. Robert Koch
The German doctor Robert Koch is considered the founder of modern bacteriology. His discoveries made a significant contribution to the development of the first ‘magic bullets’ - chemicals developed to attack specific bacteria - and Koch was awarded a Nobel Prize in 1905

9. Koch developed a new experimental method to test whether a particular micro-organism is the cause of a disease. Building on Pasteur's work on germ theory, Koch used experiments to prove that the bacterium Bacillus anthracis was the cause of anthrax. How did he do this given a need to follow a scientific method?

10. Basic Experimental Procedure
The bacterium could be observed in the tissue of anthrax victims. He extracted this bacterium from a sheep which had died of anthrax, grew it and injected a mouse with it. The mouse developed the disease as well. Koch repeated this process over 20 generations of mice, before he announced in 1876 that he had proved this bacterium caused anthrax.

11. Koch’s Postulates
1. The microorganism must be found in abundance in all organisms suffering from the disease but should not be found in healthy animals! (This process itself could be quite time consuming to isolate the given bacteria or virus.)

12. Koch’s Postulates
2. The microorganism must be isolated from a diseased organism and grown in pure culture.

13. Koch’s Postulates
3. The cultured microorganism should cause disease when introduced into a healthy organism.

14. Koch’s Postulates
The microorganism must be re-isolated from the inoculated, diseased experimental host and identified as being identical to the original specific causative agent. (Recall Koch used 20 generations of mice to prove conclusively that he had the correct bacterium.)

16. Koch Continues His Work!
Koch continued to improve his methods and techniques. By solidifying liquids such as broth with gelatine and agar, for instance, he created a solid medium for growing bacteria which was easier to handle than the liquids used by Pasteur. Koch's assistant Julius Richard Petri ( ) developed the Petri dish, which made the observation of bacteria even easier.

17. Koch and Beyond
Koch and his team also developed ways of staining bacteria to improve the bacteria’s visibility under the microscope, and were able to identify the bacterial causes of tuberculosis (1882) and cholera (1883). Adopting Koch's method, other researchers were able to identify the bacteria that caused diseases such as typhus (1880), tetanus (1884) and the plague (1894).

18. Microbes & Health (Koch’s Postulates)
A study of ‘yogurtness”

19. Microbes and Health: A study of “yogurtness”
Scientists often use model systems to simulate diseases in humans. In this activity, you will use a model to test Koch’s postulates!

20. Microbes and Health: A study of “yogurtness”
Milk will represent a healthy individual and yogurt will represent a diseased individual! At times, milk will develop into a condition that causes it to thicken and turn into yogurt; the condition will be referred to as the “yogurtness” disease!

21. Microbes and Health: A study of “yogurtness”
You will play the role of a medical investigator to determine the cause of the yogurtness. You suspect that the yogurtness disease may be caused by something that is found in the yogurt You will use Koch’s postulates to support or refute the hypothesis that microbes found in yogurt are the cause of the yogurtness disease!

22. Microbes and Health: A study of “yogurtness”
Of course, it is important to remember that this activity is a simulated investigation; real yogurt is a very healthy food. Microbes found in yogurt are harmless and do not cause disease in healthy humans.

23. Bacteria in Yogurt lactic acid bacteria are found in yogurt
Streptococcus thermophillus
lactic acid bacteria are found in yogurt
lactic acid lowers the pH in milk causing
casein (milk protein) to denature and the milk to curdle
Lactobacillus bulgaricus
Lactobacillus acidophilus
Lactobacillus casei
Bifidobacterium Bifidum
lactose pyruvic acid lactic acid

24. Procedures Overview

25. Postulate 1
The microorganism must be found in all organisms suffering from the disease, but not in healthy organisms.
Compare yogurt and milk and define the symptoms of “yogurtness”:
- microscopic observations
- textures, consistency
- smell
- pH
Milk simulates a “healthy” sample
Yogurt simulates a “diseased” sample
- When you take out a sample of the yogurt, you just take a small dap of sample from beneath the surface and you should be able to get a good sample.
When preparing the slides for the microscope, use a small amount of yogurt (like a 1:10 dilution with the drop of water). The broken down protein will block the bacteria if there is too much yogurt on the slide.
the pH of milk is 7.5 and the pH of yogurt is around 4.5
The organisms in the milk are smaller and darker when compared to the bacteria seen in yogurt. The numbers in the milk samples are usually lower than the amount of bacteria in yogurt. The bacteria you see in yogurt are round and they are usually chained together in formation of 2 or more individual bacteria. In some yogurt there are also rod shaped bacteria though not as abundant as the round ones. You will also see clumps of milk proteins (casein/curd) which is likely to take up most of the view of the slide if too much yogurt was used. This was done on a 400x microscope. You can probably see more detailed if you have access to a higher powered microscope

26. Postulate 2
The microorganism must be isolated from a diseased organism and grown in pure culture.
2. Observe the cultures using a microscope and compare the different types of colonies.
3. Inoculate 3 separate petri dishes:
Heathy individual- milk
Diseased individual- yogurt
Control bacteria- E.coli (control)
4. Grow cultures overnight at 370C
When streaking the yogurt on the LB sugar plates, very important to follow the streaking protocol to streak for single colonies. If not followed exactly, you may end up with colonies that are too small and crowded together which makes picking for individual colonies very tough.
- After streaking milk, yogurt and HB101 on the sugar LB plates and incubate them at 37 for hrs I only had colonies from the yogurt and HB101 plates. I had gotten 2 types of bacteria from some of my yogurt plates before, If you take a colony and dilute it in a drop of water, you will see round or rod shaped bacteria depending on the brand of yogurt. Usually the majority of the time I only get round shaped bacteria (individuals and in chains). The HB101 bacteria are rod shaped smaller in size when compared to the rod shaped bacteria from yogurt. If you have colonies from the milk plates, examine that too, but I have not seen any from my plates yet.

27. Postulate 3
The cultured microorganism should cause disease when introduced into a healthy organism.
5. Inoculate fresh milk with bacteria colonies from the petri dishes
6. Incubate overnight 370C
7. Assess symptoms of the subject (pH, smell, texture). Are these the same symptoms of “yogurtness”?
- After you inoculate the colonies into sterile milk and incubate for hours, you examine as you did when you look at the original yogurt sample
- After the incubation of inoculating sterile milk with yogurt bacteria and HB101 bacteria, open the tubes carefully when examining them, and beware of foul smell, especially in the HB 101 tubes and the tubes that do not appear to have become yogurt. The milk with amp should be fine.

28. Postulate 4
The microorganism must be again isolated from the inoculated, diseased experimental host and identified as being identical to the original specific causative agent
8. Observe yogurt and milk under the microscope: Can the bacteria be matched to the original culture?
Got Yogurt?