Culturing Microorganism Pure Culture - one consisting of a single type microorganism derived from a single cell - exist rarely in nature - Mixed culture : many different microorganisms living together Obtaining a pure culture 1. Sterilization 2. Isolation and cultivation of single microbial cell (production of a clone)

The 5 I’s of culturing microbes Inoculation – introduction of a sample into a container of media Incubation – under conditions that allow growth Isolation –separating one species from another Inspection Identification

Media – providing nutrients in the laboratory Most commonly used: nutrient broth – liquid medium containing beef extract & peptone nutrient agar – solid media containing beef extract, peptone & agar agar is a complex polysaccharide isolated from red algae solid at room temp, liquefies at boiling (100oC), does not resolidify until it cools to 42oC provides framework to hold moisture & nutrients not digestible for most microbes

Growing a Pure Culture Types of culture media 1. Defined media – prepared from pure chemicals, its exact chemical composition is known – has just enough ingredients to support growth is called a minimal medium 2. Complex media – made from extracts of natural materials (beef, blood, casein, yeast, and soybeans) – precise chemical composition is not known – broth : liquid complex medium cf) casein – common component of complex medium – hydrolyze with enzymes or acid to make it more soluble particially hydrolysis : protein peptide (peptone) complete hydrolysis : protein aminoacid (casein hydrolysate)

change of neutral red colors) 3. Selective media – favor the growth of particular microorganisms – used to isolate or detect the favored species in a complex mixture of other microorganisms – some media contain toxic chemicals (sodium azide, potassium tellurite, or crystall violet) – employ an extream pH value, unusual carbon source 4. Differential media – used to identify microbes by the appearance of their colonies – In blood agar : Streptococcus pyogenes  surrounded by a clear zone (lysis by red blood cells) 5. Selective-Differential media MacConkey agar – used to detect strains of Salmonella and Shigella – selective : crystal violet and bile salts (Killing of other microbes) – differential : Caliform bacteria (lactose  acid  change of neutral red colors) 6. Enrichment culture – used to isolate a particular microbes or type of micobes from a large, complex natural population ex) endospore-forming bacteria (boiling) nitrogen-fixing bacteria (nitrogen-free medium)

Table 3.4 Ingredients of a Minimal Medium Suitable for Cultivating Escherichia coli Table 3.5 Ingredients of a Minimal Medium Suitable for Cultivating Leuconostoc citrovorum

Table 3.6 Ingredients of Nutrient Broth, a Complex Medium Suitable for Cultivating Many Species of Bacteria

Types of media synthetic – contains pure organic & inorganic compounds in an exact chemical formula complex or nonsynthetic – contains at least one ingredient that is not chemically definable general purpose media- grows a broad range of microbes, usually nonsynthetic enriched media- contains complex organic substances such as blood, serum, hemoglobin or special growth factors required by fastidious microbes

Enriched media

selective media- contains one or more agents that inhibit growth of some microbes and encourage growth of the desired microbes differential media – allows growth of several types of microbes and displays visible differences among desired and undesired microbes

selective & differential media

Differential media

Miscellaneous media reducing medium – contains a substance that absorbs oxygen or slows penetration of oxygen into medium; used for growing anaerobic bacteria

The Requirements for Growth: Physical Requirements Figure 6.4

The Requirements for Growth: Chemical Requirements Carbon Structural organic molecules, energy source Chemoheterotrophs use organic carbon sources Autotrophs use CO2

The Requirements for Growth: Chemical Requirements Nitrogen In amino acids and proteins Most bacteria decompose proteins Some bacteria use NH4+ or NO3– A few bacteria use N2 in nitrogen fixation Sulfur In amino acids, thiamine and biotin Some bacteria use SO42– or H2S Phosphorus In DNA, RNA, ATP, and membranes PO43– is a source of phosphorus

The Requirements for Growth: Chemical Requirements Trace elements Inorganic elements required in small amounts Usually as enzyme cofactors

The Requirements for Growth: Chemical Requirements Organic growth factors Organic compounds obtained from the environment Vitamins, amino acids, purines, and pyrimidines

Culture Media Culture medium: Nutrients prepared for microbial growth Sterile: No living microbes Inoculum: Introduction of microbes into medium Culture: Microbes growing in/on culture medium

Agar Complex polysaccharide Used as solidifying agent for culture media in Petri plates, slants, and deeps Generally not metabolized by microbes Liquefies at 100°C Solidifies ~40°C

Culture Media Chemically defined media: Exact chemical composition is known Complex media: Extracts and digests of yeasts, meat, or plants Nutrient broth Nutrient agar

Anaerobic Culture Methods Reducing media Contain chemicals (thioglycollate or oxyrase) that combine O2 Heated to drive off O2

Anaerobic Culture Methods Anaerobic jar Figure 6.5

Anaerobic Culture Methods Anaerobic chamber Figure 6.6

Capnophiles Require High CO2 Candle jar CO2-packet Figure 6.7

Selective Media Suppress unwanted microbes and encourage desired microbes. Figure 6.9b–c

Selective Media Contains one or more agents that inhibit the growth of a certain microbe and thereby encourages, or selects, a specific microbe. Example: Mannitol Salt Agar encourages the growth of S. aureus.

Differential Media Make it easy to distinguish colonies of different microbes. Figure 6.9a

Differential Media Differential shows up as visible changes or variations in colony size or color, in media color changes, or in the formation of gas bubbles and precipitates. Example: Spirit Blue Agar to detect the digestion of fats by lipase enzyme. Positive digestion (hydrolysis) is indicated by the dark blue color that develops in the colonies.

Growth of Staphylococcus aureus on Manitol Salt Agar results in a color change in the media from pink to yellow.

Enrichment Media Is used to encourage the growth of a particular microorganism in a mixed culture. contains complex organic substances such as blood, serum, hemoglobin or special growth factors required by fastidious microbes Ex. Manitol Salt Agar for S. aureus

Enriched media

Preserving Bacteria Cultures Deep-freezing: –50°to –95°C Lyophilization (freeze-drying): Frozen (–54° to –72°C) and dehydrated in a vacuum

Culture media Plate Broth Slant Deep http://student.ccbcmd.edu/courses/bio141/lecguide/unit2/control/images/broth.jpg http://www.mushmush.nl/images/methods/working_with_agar/slant.jpg http://82.43.123.182/globalplantclinic/images/Bacteria_plate.jpg http://student.ccbcmd.edu/courses/bio141/labmanua/lab7/images/negmotility.JPG

Some vocabulary for colonies morphology Shape: round, irregular, punctiform (tiny dot) Elevation: convex, umbonate, flat, raised Color: translucent, shiny, dull, white Texture: moist, mucoid, dry (or rough)

Colonial morphology Margin- edge http://www.rlc.dcccd.edu/mathsci/reynolds/micro/lab_manual/Coloniy_morph.jpg

FYI

Bacteria motility Non motile bacteria will only be found at the site of inoculation Motile bacteria  swim around go everywhere http://www.bact.wisc.edu/bact100/Motility.jpg

Objective 3:c How to inoculate a slant Provide a solid growth surface in a tube format (take less space) Inoculate as you did for the petri plate One streak in the middle of the surface do not dig/ nor stab. Only on the surface. http://www.liddil.com/beer/culture/slant.gif

Slant observation http://www.rlc.dcccd.edu/MATHSCI/reynolds/MICRO/lab_manual/slant_patterns.jpg

Broth observation http://www.rlc.dcccd.edu/MATHSCI/reynolds/MICRO/lab_manual/broth_patterns.jpg

Uses Broth Slant Plate Deep High concentration of bacteria Space saving solid culture Plate Individual colonies Can be used to count bacteria Deep Look at motility & oxygen requirement