*5 I’s Inoculation – Implanting Microbes into or upon culture media. Tools and methods in Bacteriology *5 I’s Inoculation – Implanting Microbes into or upon culture media. -Tools used for this are : Loop – swab – needle

1.Media- artificially prepared nutrients - Bacteria Food – can be broth (soup like) in a test tube or Agar (jello) on Petri dishes

2. Culture- observable growth on/in media examples: Cloudy Broth or colony on agar 3. Culturing – inoculating microbes into media and encouraging growth 4. Contamination – unwanted microbes 5. Pure culture – only 1 type of microbe

B. Incubation placing microbes in controlled chamber with temperature set at 20C – 40C, usually at 37C (98.6F)

C. Isolation – separating bacterial species from each * Subculture use loop or swab to dip into colony and transfer to new media

D. Inspection – preparing (slides) microbes to view with microscopes

E. Identification – determining species based on shape color and other tests * microscope most important tool for this

Figure 6.8 Characteristics of bacterial colonies-overview

Culturing Microorganisms Obtaining Pure Cultures Aseptic technique Two common isolation techniques Streak plates Pour plates A colony is a population of cells arising from a single cell or from a group of attached cells 9

Streak Plate Figure 6.10a, b

Figure 6.9 Streak plate method of isolation-overview http://www.sumanasinc.com/webcontent/anisamples/microbiology/streakplate.html

Figure 6.10 Pour plate method of isolation-overview

Culturing Media A. Liquid – semisolid- solid B. Counting bacteria in 1. Broth cloudiness 2. Slide grid with microscope 3. Coulter counter

Types of media 1. General purpose –grows wide variety of microbe species 2. Selective media – this inhibits the growth of many species, but encourages certain species 3.Differential Media – Wide variety of species but each species looks different

Specialty Medias

Culture Media Complex Media/general media: Extracts and digests of yeasts, meat, or plants Nutrient broth Nutrient agar Complex polysaccharide generally not metabolized by microbes

Figure 6.11 Slant tube containing solid media Butt

Figure 6.12 An example of the use of a selective medium Bacterial colonies Fungal colonies pH 7.3 pH 5.6

Figure 6.13 The use of blood agar as a differential medium Beta-hemolysis Alpha-hemolysis No hemolysis (gamme-hemolysis)

Figure 6.15 Use of MacConkey agar as a selective and differential medium-overview

Figure 6.16 An anaerobic culture system Clamp Airtight lid Chamber Palladium pellets to catalyze reaction removing O2 Envelope containing chemicals to release CO2 and H2 Methylene blue (anaerobic indicator) Petri plates

Culturing Microorganisms Preserving Cultures Refrigeration Stores for short periods of time Deep-freezing Stores for years Lyophilization (freeze-drying): Frozen and dehydrated in a vacuum Stores for decades 23