Tools of the Laboratory:

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Tools of the Laboratory

• The Methods for Studying Microorganisms
• Chapter 3

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Chapter Overview

• Visualization and specimen handling
• Implementation of the 5 Is
• Cultures on media
• Variation in media
• Chemical
• Physical
• Functional

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Chapter Overview

• Growth and isolation of microbes for pure
  cultures for study and testing
• Cultures for microbial morphology, biochemistry
  and genetic studies
• Unknown, invisible samples become known and
  visible

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Chapter Overview

• Microscopes are vital tools in this process
• Several forms of microscopes
• Light
• Radiation
• Electron
• Specimen prep includes fresh and fixed
• Staining for highlight and contrast

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Visualization

• Paramecium seen using two types of microscopy.
• Confocal providing three dimensional images
• Fluorescent to highlight specific structures

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The 5 Is of culturing microbes

1. Inoculation introduction of a sample into a
   container of media
2. Incubation under conditions that allow growth
3. Isolation separating one species from another
4. Inspection
5. Identification

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The 5 Is of culturing microbes

• Inoculation introduction of a sample into a
  container of media

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Fig. 3.1a

• Any source for microbes
• Body fluids, tissues, foods, water or soil
• Use of sampling device swab, syringe, urine
• Transport system to hold, maintain, and preserve

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Inoculation

• Sources urine, CSF, blood, stool, fluids, wound
• Culture cultivate
• Inoculum introduction of minute sample
• Medium (pl. media) nutrient source

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Inoculation
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Laboratory Culture of Microorganisms

• Successful cultivation and maintenance of pure
  cultures of microorganisms can be done only if
  aseptic technique is practiced to prevent
  contamination by other microorganisms.

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The 5 Is of culturing microbes

1. Incubation
2. Isolation

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2. Incubation 3. Isolation
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The 5 Is of culturing microbes

1. Isolation separating one species from another

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Isolation

• Colony if an individual bacterial cell is
  separated from other cells has space on a
  nutrient surface, it will grow into a visible
  mound of cells
• A colony consists of one species

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Isolation

• A pure culture contains only one species or
  strain
• A colony is a population of cells arising from a
  single cell or spore or from a group of attached
  cells
• A colony is often called a colony-forming unit
  (CFU)

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Isolation technique

• Stages in the formation of an isolated colony
  showing the microscopic event and the macroscopic
  result. Separation techniques such as streaking
  can be used to isolate single cells. After
  several numerous subdivisions a colony will form.
  This is a simple way to separate mixed samples.

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Fig. 3.3

1. Quadrant smear steps
2. Isolated colonies
3. Loop dilution
4. Plate appearance
5. Spread plate
6. Result

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Streak Plate
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Streak Plate
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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

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• Figure 3.4
• Sample liquid media
• Flows when container tilted
• Enterococcus faecalis selective media

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Figure 3.5 Sample semisolid media

• Clot-like soft consistency, ½ agar
• SIM medium
• Sulfur
• Indole
• Motility

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Fig. 3.6 Solid Media

1. 1-5 agar maintains solid consistency even at
   incubation temperatures
2. Gelatin media can be liquefied if organisms
   produce enzymes that digest the protein

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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

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Enriched media

1. Blood agar plate throat culture
2. Chocolate agar Neisseria gonorrhoeae

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• 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

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selective differential media
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Selective media

• Mannitol salt isolate Staphylococcus
• MacConkey for differentiation of lactose
  fermenters
• () pink-red
• (-) off white

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Differential media

• TSI 3 carbohydrates, phenol red, and iron for
  H2S (reactions L-R)
• No growth
• Growth, no acid
• Acid in butt
• Acid throughout
• Acid butt, H2S
• CHROMagar for urinary pathogens

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Miscellaneous media

• reducing medium contains a substance that
  absorbs oxygen or slows penetration of oxygen
  into medium used for growing anaerobic bacteria
• carbohydrate fermentation medium- contains sugars
  that can be fermented, converted to acids, and a
  pH indicator to show the reaction basis for
  identifying bacteria and fungi

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Carbohydrate fermentation media
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Fig. 3.12 Various conditions of cultures

1. TSA (L-R) Escherichia coli (white), Micrococcus
   luteus (yellow), Serratia marcescens (red)
2. Mixed culture M. luteus / E. coli
3. Contaminated note white colony

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Fig. 3.1d