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

3. Microbes and Health Kit – Core Content Alignment
Scientific Inquiry
Interpretation of experimental results
Use of experimental controls
Evaluation of hypothesis
Microscopy
Cell and Molecular Biology
Bacterial metabolism
Prokaryotic cell structure and cell division
Effects of temperature and pH on bacterial growth
Antibiotics
Chemistry of Life
Effects of pH on proteins
Enzymes
Protein structure and function
Environmental and Health Science
Epidemiology and disease
Microbiology
Evolution
Adaptation to environment
Bacterial defense mechanisms
Genetics
Variation in bacteria

4. Workshop Time Line Introduction
Preparation of microscope slides. Observe cultures and asses disease symptoms (pH, smell, texture)
Isolate disease causing pathogens and grow in pure culture ( grow on LB sugar plates)
Inoculate milk samples
Assess disease symptoms (pH, smell, texture) from pre-inoculated milk samples and compare to the original bacteria
Laboratory Extensions
Timeline of lab
Postulate 1
Assess disease symptoms: pH, smell, texture
Demonstration of yogurt bacteria on slide with projector- include considerations for making the slides
Postulate 2
Isolate disease causing pathogens
Spread yogurt, milk and E.coli for single colonies on LB Sugar plates
Julia Childs pregrown plates
Demonstration of bacteria on slide with projector
Postulate 3
Inoculate 5 tubes of milk with bacteria
Julia Childs preinoculated milk
Postulate 4
Demonstration of yogurt bacteria on slide with projector

5. What can you teach with the Microbes and Health Kit?
Practice sterile microbial techniques commonly used in research
Study the role of microbes in disease and health
Learn how cells metabolize nutrients to form other products
Utilize Koch’s Postulates to identify the causative agent for disease
Your students will attempt to discover the causative agents that turn milk into yogurt

6. Mammalian Cell Culture
4.2 Sources of DNA
In nature, DNA is made in cells. While the basic DNA molecule is the same between organisms the packaging and location within the cell vary.
Medium: nutrient source used to grow cells in a lab.
Mammalian Cell Culture
Growing mammalian cells in culture is more challenging than growing bacterial cells
Mammalian cells are grown in a broth culture
Lysis: process by which cells are broken open and release their DNA
Viral DNA
Viruses are classified according to the type of cell they attack:
Bacterial (bacteriophages)
Plant
Animal

7. Prokaryotic DNA : no nucleus or membrane bound organelles (ex
Prokaryotic DNA : no nucleus or membrane bound organelles (ex. Bacteria – E. coli. DNA is floating in the cytoplasm. Bacteria typically contain only one, long, circular DNA molecule (chromosome). E. coli, has 1 chromosome with ~4000 genes and 4.6 million bp. The entire genome codes for RNA and proteins.
Plasmid = extra small ring of DNA floating in the cytoplasm (5-10 genes). (R plasmids – contain the antibiotic resistance genes.

8. Bacteria can transfer plasmids (ex
Bacteria can transfer plasmids (ex. For antibiotic resistance = evolving).
Transformed bacteria = scientists transfer “genes of interest”
DNA vectors = piece of DNA that carries one or more genes into a cell (plasmids) – insulin

9. Bacterial Operon. An operon contains the controlling elements that turn genetic expression ON and OFF.
Promoter – region at the beginning of a gene where RNA polymerase binds

10. Bacterial Cell Culture

11. Bacterial Cell Culture – grown in a mixture of water and protein media broth: suspensions of millions of floating cells. Reproduce quickly since they have better access to nutrients than colonies on a plate. (replicate ~ every 20 min). Agar – powdered agar is mixed with water and sterilized and poured into Petri dishes to solidify in 15 – 20 min.

12. Media Preparation & Sterile Technique
Sterile technique – process of doing something without contamination by unwanted organisms or their spores. (ex. Surgery , media preparation).
Autoclave – instrument that creates high temperature and pressure to sterilize equipment and media.

13. Laminar flow hood – decreases chances of contamination by filtering microorganisms from the air.

14. Sterile Technique – for Yogurtness Lab
When culturing bacteria it is important to avoid contamination. Contaminating bacteria and
molds are found everywhere, including on hands and lab benchtops, so it is important to avoid
these surfaces. The round circle at the end of inoculating loops and the surfaces of agar plates
should not be touched or placed onto potential contaminating surfaces. Wipe down lab benches
with 70% alcohol or a 10% bleach solution ( 1 hour soak) wearing appropriate safety equipment.
WASH HANDS!!!

15. Bacteria, Bacteria, Bacteria
The single most successful life form on earth – greatest biomass.
Prokaryotic organisms
Exist in soil, water, in and on animals, plants and humans
Several distinct morphologies
coccus – spherical, bacillus – rods, spiral forms
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 -)
5,000 – 50,000 in a row = 1 inch!
Bacteria, Bacteria,
Bacteria
Keep slide in ppt but do not plan to present

16. 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

17. 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
-acidic condition inhibits growth of other microorganisms that might spoil yogurt.
Lactobacillus bulgaricus
Lactobacillus acidophilus
Lactobacillus casei
Bifidobacterium Bifidum
lactose pyruvic acid lactic acid
Probiotics – these organisms produce yogurt
Anaerobes – anaerobic respiration / fermentation

18. 2. Cell Walls – way to differentiate bacteria
Types are detected using Gram Staining
Purple = Gram positive  has peptidoglycan
GRAM-POSITIVE BACTERIA HAVE A THICKER LAYER OF PEPTIDOGLYCAN IN THEIR CELL WALLS, MADE OF A PROTEIN-SUGAR COMPLEX THAT TAKES ON THE PURPLE COLOR DURING GRAM STAINING
Can be treated with antibiotics
Pink = Gram negative  has 2nd outer layer that absorbs only red stain
GRAM-NEGATIVE BACTERIA HAVE AN EXTRA LAYER OF LIPID ON THE OUTSIDE OF THE CELL WALL AND APPEAR PINK AFTER GRAM STAINING
Likely to resist antibiotics

20. Antibiotics and Drug Resistance
Anti-bacterial antibiotics are one of the main theraputic tools to control and treat many bacterial infectious diseases. These may be:
- Bactericidal – Kill bacteria
Bacteriostatic – prevent bacteria from dividing
Antibiotics have various modes of action
- May inhibit important bacterial enzymes
- May destroy cell wall components
Antibiotic Resistance
- Due to overuse/misuse of antibiotics
- Some bacterial strains develop resistance as an outcome of natural selection pressures
Antibiotics
and Drug Resistance
Genetic Transfer
Genetic material can be transferred between bacteria by several means, most often by:
Conjugation
Transformation
And, Transduction

21. 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.

22. 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.

23. Procedures Overview

24. Laboratory Quick Guide
- There is a limited amount of loops so don’t use them for anything else unless the manual asks you to use them.

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.
- 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.
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”?

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?

29. Laboratory Extensions
Culture microbes from anywhere
- Surfaces
- Pets
- Homes
- School
- Water
Study the use of antibiotics
Grow liquid culture to teach
- Bacterial growth curves
- Serial dilutions
- Counting bacteria
- Spectrophotometry

30. -Enhancer – a section of DNA that increases the expression of a gene
Eukaryotic DNA
-protists, fungi, plants, & animal cells
-same bases (A,T,C,G)
-H bonds link bases
-DNA packaged in chromosomes (most eukaryotes have several chromosomes / cell)
-Humans = 46 chromosomes / somatic cell (23 pairs)
-Humans = 23 chromosomes / gamete (NO PAIRS)
-much eukaryotic DNA is noncoding (does not transcribe into a protein)
-genes that are far apart involved in recombination
Eukaryotic Gene. Eukaryotic genes have a promoter to which RNA polymerase binds, but they do not have an operator region.
-Enhancer – a section of DNA that increases the expression of a gene
-Intron – the region on a gene that is transcribed into an mRNA molecule but not expressed in a protein
-Exon – the region of a gene that directly codes for a protein; it is the region of the gene that is expressed

31. -Transcription factors – molecules that work to either turn on or off the transcription eukaryotic genes
-Histones – nuclear proteins that bind to chromosomal DNA and condense it into highly packed coils

32. Mammalian Cell Culture
-more challenging than growing bacterial cells
-grown in broth cultures in fermenters

33. Glycoprotein Enveloped Virus – flu and AIDS
Viral Structure (Helical Virus)
-tobacco mosaic virus
Viral DNA
-not living
-require a host
-protein + nucleic acid
25 – 250 nm (smaller than bacteria!)
Nonpathogenic – not known to cause disease
Classified by shape
Polyhedral
Helical
Glycoprotein enveloped
Viral Structure (Polyhedral Virus) - papilloma – warts/ cold virus
Glycoprotein Enveloped Virus – flu and AIDS

34. Viral DNA
3 Types of Viruses:
Bacterial (bacteriophages)
Plant
Animal
Bacteriophages – viruses that infect bacteria
Gene therapy – the
process of treating a
disease or disorder by
replacing a dysfunctional
gene with a functional
one (viruses are often
used to carry a normal
gene into cells).
Ex. Parkinson’s, cystic fibrosis, diabetes, & some cancers

35. Recombinant DNA Technology 4.3 Isolating and Manipulating DNA
Genetic Engineering modifications of DNA code)Requires:
Identification of the molecule(s)
Isolation of the instructions (DNA sequence/genes) for the production of the molecule(s)
Manipulation of the DNA instructions
Harvesting of the molecule or product, testing it, and marketing it
Methods to create new DNA molecules – Ex. rhInsulin, rhuman growth hormone, r gamma interferon, rHER2 antibody, rrennin = chymosin, rt-PA (activase) to dissolve blood clots (grown in CHO cells in broth culture)
Site-Specific Mutagenesis
Process of including changes (mutagenesis) in certain sections (site-specific) on a particular DNA code (may stunt or inhibit product production)
Gene Therapy – see previous notes