1. Bacterial Culture Media First Semester 2016-2017
Faculty of Medicine and Health Sciences
Microbiology Lab
Experiment 2
Bacterial Culture Media First Semester

2. Brief history:
Upon discovery of bacteria, it was important to develop methods to culture bacteria in the lab in order to:
1- Study them and
2- Carry out experiments.
So, it was important to develop culture media that support the growth of bacteria.
Initially, culture media were very simple:
Louis Pasteur used simple broths made up of meat extracts.
Robert Koch realized the importance of solid media and used potato pieces to grow bacteria.
It was on the suggestion of Fannie Eilshemius, wife of Walther Hesse (who was an assistant to Robert Koch) that agar was used to solidify culture media.

3. Chemical (Nutritional) Requirements for Bacterial Growth:
Every living organism must have in its environment all of the substances required for:
1- Energy generation
2- Biosynthesis of molecules required for survival and reproduction/replication.
3- Appropriate physical conditions
All chemicals and elements utilized for bacterial growth and are referred to as nutrients.

4. Nutrients required for bacterial growth can be classified as:
1- Major Elements: are those needed in large quantities

5. 2- Trace Elements: are metal ions that are required for bacterial growth in very small amounts such as Mn, Co, Zn, Cu, and Mo. (They are usually found in water). These ions are usually needed co-factors for certain enzymes
Artificial culture media use for bacterial growth provide all the essential nutrients required for bacterial growth.

6. Growth factors:
Any types of nutrients that are required that is required for the growth of a particular bacterium because it cannot be synthesized by this bacterium.
The inability of certain bacteria to synthesize such nutrients is attributed to the fact that:
1- The bacteria do not have the metabolic pathway required for their production or
2- the metabolic pathway required for their production is blocked by mutation (s).
In most cases, growth factors required for growth could be:
One or more purine and/or pyrimidine nucleotide(s): needed for nucleic acid synthesis
One or more amino acid(s): needed for protein synthesis
One or more vitamin(s): needed as coenzymes and functional groups for certain enzymes

7. Nowadays, culture media are used not only for bacterial growth, but also for:
Transport
Diagnosis
Storage
Despite all the development that the scientists have approach regarding the invention of culture media, these are still certain types of bacteria that are:
1- So far unculturable (such as Treponema pallidum, Mycobacterium leprae ). Such kinds of bacteria are propagated in animals
2- Certain bacteria are obligate intracellular: these bacteria are just like viruses, they can be only grown in living cells ( Examples: Chlamydia, Rickettsia and Coxiella ). Such bacteria are cultured in cells

8. Media Classification: Bacterial culture media can be classified according to its chemical composition, physical nature and intended usage

9. Types of Media based on their chemical composition:
1- Chemically-defined (synthetic) media: (minimal medium):
The exact chemical composition is known
Provide only the exact nutrients (including any growth factors) needed by a bacterium for its growth.
Such kind of media are used currently for research purposes to study the exact chemical requirements needed for the growth of a particular bacterium
2- Complex (undefined) media:
The exact chemical composition is unknown.
Such media contain complex materials of biological origin so that they are rich in nutrients such as milk , yeast extract or beef extract.
So, complex culture media are very rich in nutrients that support the growth of many types bacteria ( Just like Open Buffet)
Such kind of culture media are more practical for routine couture processes

10. Complex culture media can be classifies based on their function:
Supportive media:( General purpose media): contain nutrients that support the growth of most non-fastidious bacteria without giving any particular bacteria growth advantage ( Example: nutrient agar and nutrient broth)
Enriched media: General purpose media supplemented by blood or other special nutrients to support the growth of fastidious organisms ( Example: Blood agar)
Selective media: Support the the growth of certain bacteria, while inhibiting others. Examples
Mac Conkey agar: it contains bile salts and crystal violet that inhibit the growth of most Gram- positive bacteria. Thus it allows only the growth of Gram-negative bacilli
Eosin methylene blue agar (EMB): it is a selective medium allows the growth of only Gram- negative bacilli. Eosin and methylene blue inhibit the growth of most Gram positive bacteria. Thus, this medium allows the growth of Gram-negative bacilli (similar to Mac Conkey agar)
Columbia agar: it contains colistin and nalidixic acid antibiotics that allow the growth of Gram- positive bacteria but inhibit the growth of Gram-negative bacteria.

11. Differential media: are complex media that distinguishes one microorganism from another growing on the same media using  biochemical characteristics of a microorganism.
Examples
Salmonella Shigella Agar (SS agar: it is differential medium widely used to isolate Salmonella and Shigella from clinical samples.
Mannitol Salt Agar (MSA): is a differential medium that has a high concentration of salt (7.5%). This salt concentration allow the growth of Staphylococci but inhibit the growth of most other bacteria that are inhibited by the high salt concentration.
Note: Selective media and enriched media can be also differential for certain characteristics of bacteria, so some of these media may be called selective and differential

13. What is agar Agar?:
Is a sulfated polysaccharide that is used to solidify liquid media
Most bacteria cannot degrade it. So it can be sued only as a solidifying agent
Agar melts at 85 °C and solidifies between °C 
Before agar, microbiologist tried to used gelatin as a solidifying agent but, it was not a good choice because:
Gelatin melting temperature is not suitable at the incubation temperatures used to grow bacteria ( it may melt at these temperatures)
Many bacteria can degrade gelatin and used is as a nutrient

14. Physical and Environmental Requirements for Bacterial Growth
1- Oxygen Requirements:
Based on Oxygen requirement, bacteria may be classified as:
Obligate aerobes require O2 for growth; they use O2 as a final electron acceptor in aerobic respiration
Obligate anaerobes: they do not need or use O2 . In fact, O2 is a toxic substance, which either kills or inhibits their growth. Obligate anaerobic bacteria produce energy by either by certain methods such as fermentation and anaerobic respiration
Facultative anaerobes (or facultative aerobes): these are organisms that can switch between aerobic and anaerobic types of metabolism. Under anaerobic conditions, they grow by fermentation or anaerobic respiration, but in the presence of O2, they switch to aerobic respiration.
Aerotolerant anaerobes: these are bacteria with an exclusively anaerobic but they are not sensitive to the presence of O2
Microaerophilic: need O2 in low concentration

15. Oxygen Utilization and Free Radicals:
Utilization or exposure to O2 results in the generation of highly reactive and toxic molecules known as free radicals such as superoxide (O2.-) and Hydrogen peroxide (H2O2).
Unless the cell is able to neutralize these molecules, free radicals oxidize various cellular molecules to inactivate them resulting in major cell damage or cell death.
In aerobic bacteria, facultative- anaerobic bacteria, microaerophilic and aerotolerant bacteria, the lethal accumulation of free radicals is prevented by the presence of an enzymatic systems:
Superoxide dismutase
Catalase (or peroxidase).
Obligate anaerobes lack such enzymes and thus upon exposure to O2, the generated free radical molecules may kill the anaerobic bacteria or at least inhibit their growth.

16. Superoxide Dismutase, Catalase and Peroxidase:
These enzymes detoxify oxygen free radicals that are generated by cells in the presence of O2.
The distribution of these enzymes in cells determines their ability to survive in the presence of O2.

17. 2- pH Requirements:
Based on the optimum pH needed fore their growth, Bacteria can be classified as:
Acidophils: are bacteria that grow at an optimum pH well below neutrality (7.0)
Neutrophils: are bacteria that grow best at neutral pH
Alkaliphils: are bacteria that grow best under alkaline conditions.

18. 3- Temperature requirements: Based on the optimum temperature needed for their growth, bacteria may be classified as follows:
Bacteria with an optimum temperature near 37°C are called mesophiles.
Bacteria with an optimum temperature between about 45°C and 70°C are thermophiles.
Some bacteria have an optimum Temperature of 80°C or more. These bacteria are called extreme thermophiles or hyperthermophiles, respectively
Example: Some Archaea bacteria are able to grow at a temperature 115C.
Some bacteria are able to grow at low temperature. Such kind of bacteria are called psychrophiles. They can grow at a temperature as low as 0°C.

20. 4- NaCl Requirement (ionic concentration):
The most common solute in nature that occurs over a wide range of concentration is salt (NaCl).
Microorganisms can be classified based on NaCl requirement for growth as:
1- Halophiles: are bacteria that require NaCl for growth. Halophiles can be classified as:
Mild halophils: require 1-6% salt concentration for growth
Moderate halophils: require 6-15% salt concentration for growth
Extreme halophils: require 15-30% salt concentration for growth.
2- Halotolerants: these are bacteria that do not need NaCl for growth but can withstand a moderate range of salt concentrations.
3- Nonhalophils: these are bacteria that do not need salt for growth and are very sensitive to salt (they are killed by NaCl)

21. 5- Osmolarity and Water Requirement:
Osmolarity is determined by solute concentration in solutions.
That is to say, as solute concentration (Osmolarity) increases, water concentration decreases and vise-versa (( Increased solute concentration means increased Osmolarity and decreased water activity (Aw))
Water concentration in solution is measured as water activity (Aw). The value of water activity of pure water 1
As solute concentration increases in solution, water activity is becoming less that 1
So, in solutions, as solute concentration increases, osmolarity increases, but water activity (concentration) decreases.

22. Osmophils: these are bacteria that can live in the presence of high sugar concentration (high osmotic pressure) or it is possible to say environments that are low in water activity (Aw).
Xerophils: Organisms that can live in almost dry environments

23. Preparation of bacterial culture medium:
Culture media these days are prepared by specialized companies
Most culture media come as powder
A- Preparation of Agar media:
The preparation process is carries out according to the manufacturer instructions:
1- Use clean glass wares (flasks) ( rinsed with demonized water)
2- Dissolve so and so gram in certain volume of distilled or demonized water
3- Boil to dissolve
4- Close the opening of the flask with cotton blogs and cover the cotton blogs with aluminum foil
5- Place a small piece of autoclaving tape on the flask

24. 6- Sterilize the culture medium by Autoclaving for 15-30 min
7- Allow the medium to cool down ( 50C)
8- Pour the medium into sterile Petri dishes under a septic condition
Septic condition implies:
Clean the bench with alcohol
The lab must have no air current ( windows and doors are closed )
You must work beside the Benson burner
The hair must be covered
Do not talk while pouring the medium
9- Allow the plates to solidify and then place them ( up side down) in the incubator for over night (this will allow the detection of any contamination)
10- Next day, place the plates in plastic bags, write on the bag the name of the medium and the date of preparation
11- Store the plates in the fridge

25. Notes: 1- For Blood Agar Preparation:
Allow the autoclaved medium to cool down ( 50 C)
Add sheep blood to the medium at a concentration of 5%
2- For Chocolate Agar Preparation:
Add sheep blood to the medium at a concentration of 5% while the medium is still hot
High temperature causes rupture of red blood cells and oxidation of hemoglobin. Oxidized hemoglobin has a brown color, hence the name chocolate agar

26. B- Preparation of Broth media ( liquid media):
The preparation process is carries out according to the manufacturer instructions:
1- Use clean glass wares (flasks) ( rinsed with demonized water)
2- Dissolve so and so gram in certain volume of distilled or demonized water
3- Boil to dissolve ( if needed)
4- aliquot the broth in clean 10 ml glass tubes ( fill half of the tube)(the volume of the used tubes is according to what needed)
5- Close the opening of the tubes with cotton blogs and cover the cotton blogs with aluminum foil
6- Place a small piece of autoclaving tape on the tubes

27. 7- Sterilize the filled tubes by Autoclaving for 15-30 min
8- Allow the tubes to cool down to room temperature
9- Store the tubes in the fridge

28. Culture Media For Fungi Example: Sabouraud Dextrose Agar (SDA) It is a culture medium that promotes the growth of medically important fungi (yeast and molds), but inhibits the growth of many bacteria and many environmental fungi. Inhibition of the growth of many bacteria and many environmental fungi is achieved by the addition of : 1- Inhibition of bacteria: by the addition of certain antibiotics such as chloramphenicol 2- Inhibition of environmental fungi: by the addition of cyclohexamide

29. For more detailed and interesting reading about culture of fungi follow the link: