Microbiology: Principles and Explorations Sixth Edition Chapter 6: Growth and Culturing of Bacteria Copyright © 2005 by John Wiley & Sons, Inc. Jacquelyn G. Black

Some microorganisms are autotroph : synthesis there own amino acids. Other micro organisms need to supply them by amino acids and vitamins.

Types of Culture Media Natural Media: In nature, many species of microorganisms grow together in oceans, lakes, and soil and on living or dead organic matter Synthetic medium: A medium prepared in the laboratory from material of precise or reasonably well- defined composition (from carbon source, nitrogen source, amino acids and vitamins ) Complex medium: contains reasonably familiar material but varies slightly in chemical composition from batch to batch (e.g. peptone, a product of enzyme digestion of proteins)

The microorganisms that can live in a synthetic media differs in their needed to the substance : 1- microorganisms that needs a lot of substance to grow, we call it (fastidious) متطلب ex. Salmonella

Commonly Used Media Yeast Extract ( sublimit as source for vitamins and amino acids) Casein Hydrolysate ( good source for proteins, used as a sublimit ) Serum ( contains protein vitamins and amino acids) Blood agar ( very good media for the isolation of fastidious microorganisms, has a lot of nutrients and used for the diagnoses by knowing that certain microorganisms capable to hemolysis ex. Gram +ve bacteria streptococcus pyogenes )

Chocolate agar ( it made of hydrolysis blood by heating and this media also support the growth of fastidious microorganisms )

Diagnostic medias : Selective, Differential, and Enrichment Media Selective medium: ( usually we put anti microbial agent then just the resistant microorganisms (gram –ve bacteria) remain and the susceptible one(usually gram +ve bacteria) die) encourages growth of some organisms but suppresses growth of others (e.g. antibiotics such as sulfadiazine and polymyxin sulfate media SPS and MacConkey agar ) Differential medium: (ex. Blood agar ) contains a constituent that causes an observable change (e.g. MacConkey agar and SPS)

MacConkey agar have crystal violet so its going to kill gram +ve bacteria, select the gram –ve and it differentiate between the gram –ve bacteria, the lactose fermental from non lactose fermental Lactose fermental: gives lactic acid and appears in red color ( the media have pH indicator) Non lactose fermintation: no color Usually the microorganisms that lives in the GI tract of human being are lactose fermenters and which lives in animals non lactose fermenters.

We use the MacConkey agar to differentiate between Escherichia coli (lives in the GI tract – lactose fermenter-) and the Salmonella causes the salmonellosis مرض من الميونيز( lives in animals- non lactose fermenter-) SPS: it differentiate based on the production of hydrogen sulfite and used for clostridium botulinum -that causes the botulisms in the canned food – and its appear in black color

Enrichment medium: contains special nutrients that allow growth of a particular organism that might not otherwise be present in sufficient numbers to allow it to be isolated and identified Ex. Selenium and Salmonella we put the selenium to enhance the growth of Salmonella Chromogenc agar are differential agar.

Three species of Candida can be differentiated in mixed culture when grown on CHROMagar Candida plates Red color refers to the lactose fermentation

Identification of urinary tract pathogens with differential media (CHROMagar)

Don’t Leave Home Without Your CO 2

Oxygen Obligate aerobes Obligate anaerobes Microaerophiles Capnophiles Facultative anaerobes Aerotolerant anaerobes

Candle Jar culture of anaerobes and microaerophiles

Broth culture for the anaerobes : we put some agar to make the media relatively rigid and we put SH- group like (reducing agent) thioglycolate and sistine to capture the oxygen. if I want make it in agar ….. in the past: they cultured the anaerobe in a candle jar, the CO2 that produced from thre candle is useful for the microaerophiles. Recently : they use an anaerobic jars with gas batches to produce CO2 or N2.

To culture obligate anaerobes, all molecular oxygen must be removed and kept out of medium. Agar plates are incubated in sealed jars containing chemical substances that remove oxygen and generate carbon dioxide or water

Anaerobic Transfer Anaerobic cabinet in the diagnostic labs

In the aerobes some time the microorganisms need a lot of air so we provide the culture with filtered air (by filtrating the air by air filters to prevent any contamination)

Preserved Cultures To avoid risk of contamination and to reduce mutation rate, stock culture organisms should be kept in a preserved culture, a culture in which organisms are maintained in a dormant state 1.Lyophilization : to eliminate the water from culture (powder) by a Frees Dryer. Used for Long term maintenance 2.Frozen at -70 o C : with 10% glycerol. Used for Long term maintenance. 3.Refrigeration: its not so good. Used for short term maintenance.

Stock culture: it’s the main culture and I take subculture medias from it (subculturing) Aseptic conditions : Sterile gloves, work beside the flame …etc To reduce the contamination. We cant make subcultures from the subculture for many times (maximum 4 subcultures) because it may cause a mutation so we have to retained to the stock culture.

Reference culture (type culture): a preserved culture that maintains the organisms with characteristics as originally defined. example for companies that have reference culture : ATCC American type culture collection.

For the diagnosis of the microorganisms we have two methods : 1- biochemical tests.(phenotypic characterization) Currently we use something called multiple diagnostic tests ex. In slide molecular tests. (genotypic characterization, and its more accurate)

Methods of Performing Multiple Diagnostic Tests 1.The Enterotube System 2.The Analytical Profile Index (API) System

The Enterotube Multitest System

Based on it we take a number and this number determine the microorganism

The API System: Various species of Enteobacteriaceae are shown here with differences in reactions that enable them to be identified ATCC sample = reference culture of microorganisms