Fundamental Techniques in Microbiology Dr Paul D. Brown paul.brown@uwimona.edu.jm BC10M: Introductory Biochemistry

Fundamental Techniques Microscopy Staining Aseptic technique Sterilization and waste disposal Media preparation

Microscopy Measurement Microorganisms are very small Use metric system Metre (m) : standard unit Micrometre (m) = 1 x10-6 m Nanometre (nm) = 1 x10-9 m Angstrom (Å) = 1 x10-10 m

Terms Relevant to Microscopy For MICR 2909 Lecture 2, 2001 Terms Relevant to Microscopy Total Magnification Eyepiece x objective lens Resolution Ability of the lens to distinguish two points as separate Optimal RP achieved with blue light Theoretical limit for light microscope is 0.2 m Refractive Index (η) Measurement of relative velocity at which light passes through a material. η= 1.0 in air η (Oil) = η (glass) = up to 1.5 R.P. = 0.5 (lambda) / (η sinØ) BSc(MolBiol) Lect 2.ppt

Resolving Power Optical Instrument Resolving Power R.P. in Angstroms Human eye 0.2 mm 2,000,000 Å Light microscope 0.2 m 2000 Å Scanning electron microscope 5-10 nm 50-100 Å Transmission electron microscope 0.5 nm 5 Å

Types of Microscopes Simple: one lens Compound: more than one lens

The Compound Microscope READ BOTTOM TO TOP! enters the eye sees virtual, inverted image further magnif. by ocular forms magnified real image enters objective focuses light on object light enters condenser ocular objective object condenser

Objectives 10X Scanning Find the object 40X High-Dry Focus the object 100X Oil immersion Fine focus (Course focus) (Fine focus)

The Condenser Functions Height of condenser controls Focus light on object plane Ensure adequate intensity Height of condenser controls Uniformity of brightness Contrast (minimises “stray light”) (Indirectly) angle of light entering objective

Condenser Height correct too low stray light area  . 1 . intensity angle correct too low

Use of Immersion Oil R decreased     NA =  sin  = 1,  > 41 , rays reflected inside glass (critical angle) max NA = 0.65 critical angle = 90, wide rays enter objective max NA = 1.5 = 1, Air = 1.5, Immersion oil R decreased     NA =  sin 

Condenser Iris Diaphragm  aperture diaphragm wide aperture  large , large NA, low R (good resolution), poor contrast (if too wide) narrow aperture  small , small NA, high R (poor resolution), good contrast

Bright-field Microscope Contains two lens systems for magnifying specimens Specimens illuminated directly from above or below Advantages: convenient, relatively inexpensive, available Disadvantages: R.P 0.2 m at best; can recognize cells but not fine details Needs contrast. Easiest way to view cells is to fix and stain.

Different magnifications

Special Microscopy Applications For MICR 2909 Lecture 2, 2001 Special Microscopy Applications Dark Field Phase Contrast Fluorescence Electron Microscope BSc(MolBiol) Lect 2.ppt

Dark Field Microscopy special condenser diaphragm diffracted light special condenser diaphragm occludes direct light, passes wide angle light angle too wide to enter objective diffracted light scattered enters objective objects light on dark background

Phase Contrast Microscopy For MICR 2909 Lecture 2, 2001 Phase Contrast Microscopy light rays through objects of different   change in phase, not intensity special ring-shaped condenser diaphragm special glass disc in objective change phase differences to intensity differences can view transparent objects as dark on light background (without staining) Right; human brain glial cells Uses of darkfield and Phase contrast microscopy View unstained cells Not subject to shrinkage, artefacts Some bacteria do not stain easily, e.g., spirochetes (Borrelia, Leptospira, Treponema) View living cells Can observe processes, e.g., motility, predation BSc(MolBiol) Lect 2.ppt

Fluorescence Microscopy For MICR 2909 Lecture 2, 2001 Fluorescence Microscopy Illuminate specimen with UV  visible fluorescence (filter removes harmful UV) View auto-fluorescent objects (e.g., chloroplasts) Stain with specific fluorescent dyes, which absorb in region 230-350 nm & emit orange, yellow or greenish light Images appear coloured against a dark background BSc(MolBiol) Lect 2.ppt

Electron Microscopy

Stains and Staining Bacteria are slightly negatively charged at pH 7.0 Basic dye stains bacteria Acidic dye stains background Simple stain Aqueous or alcohol solution of single basic dye

Simple Stains

Differential Stains Gram stain Crystal violet: primary stain Iodine: mordant Alcohol or acetone-alcohol: decolourizer Safranin: counterstain Gram positive: purple Gram negative: pink-red Staphylococcus aureus Escherichia coli

Gram stain – distinguishes Gram+ from Gram - Gm(+) and Gm(-) both take up CV-I equivalently CV-I is not readily removed from Gm(+) due to the reduced porosity of the thick cell wall CV-I is readily removed from Gm(-) thin peptidoglycan due perhaps to the discontinuities in the outer membrane structure introduced during the decolorization step. -removal of the cell wall (with lysozyme) from a Gm(+) bacterium results in a Gm(-) stain profile

For MICR 2909 Lecture 2, 2001 BSc(MolBiol) Lect 2.ppt Differential Stains Acid-fast stain Used to detect Mycobacterium species Acid fast stain Acid fast stain (Ziehl-Neelson) for identifying mycobacteria The lipid mycolic acid (from mycobacteria) is the determinant of retaining the basic fuchsin in the acid-fast stain BSc(MolBiol) Lect 2.ppt

Special Stains Capsule stain Klebsiella pneumonia

Special Stains Flagella stain

Special Stains Spore stain (Schaeffer-Fulton) Bacillus subtilis

For MICR 2909 Lecture 2, 2001 BSc(MolBiol) Lect 2.ppt Aseptic Technique First requirement for study of microbes pure cultures, free of other microbes Maintain a clean environment; work close to the flame Aseptic Transfers Purpose Transfer of microbiological cultures from one medium to another sterile medium without contamination of the culture, sterile medium, or the surroundings.   Principle Certain techniques are necessary to handle tubes of media, plated media, and inoculating loops or swabs. Practice in the manipulations, while maintaining aseptic conditions are known as "aseptic techniques." Refer to your laboratory text for acceptable methods of transferring microbial cultures. However, practice is the only way to master the techniques. Additional Information 1. Gather all the necessary materials (bacterial stock culture, growth media, bunsen burner, transfer tools). 2. Label the tubes properly, following the outlined instructions. 3. Check each organism to be transferred, using the stock cultures. 4. Practice adjusting the flame of the Bunsen burner. 5. Discard contaminated materials properly, referring to the guidelines given in class. Sterilize the loop or needle by holding in the flame of a Bunsen burner. The metal must glow red before sterilization is considered complete. When transferring organisms, it is important to maintain a clean environment. Talking, coughing or sneezing should be avoided when performing bacterial transfers from one media to another. Avoid drafts. Maintain a clean environment by disinfecting tabletops before and after working with microorganisms. Common surface disinfectants are 70% Ethanol and 10% Lysol. BSc(MolBiol) Lect 2.ppt

Streak plate method of isolation For MICR 2909 Lecture 2, 2001 Streak plate method of isolation At all costs, there must be prevention of contamination. Often use cotton wool stoppers to flasks, tubes. Petri dish: ideal for solid medium and allows gaseous diffusion without dust Development of solid media needed for colony formation initially used surface of freshly cut vegetables eg potato gelatin (1881) used low melting point (< 37oC) protein, therefore a nutrient agar-agar (Hess, Koch’s lab) polysaccharide - nutritionally inert melts 100oC ; solidifies ~40oC Streak Plate Method of Isolation Purpose The streak plate technique is the most widely used method of obtaining isolated colonies from a mix of cultures.   Principle The streak plate technique is essentially a method to dilute the number of organisms, decreasing the density. This allows for individual colonies to be isolated from other colonies. Each colony is considered "pure," since theoretically, the colony began with an individual cell.  Additional Information (see also p. 53 in the lab text for diagrams.) 1. Begin with inoculating the first, or primary, quadrant of the agar plate. Use a light touch. Don't penetrate or scrape the agar surface. Cover plate with lid. 2. Flame the loop, cool by touching an uninoculated portion of the surface. 3. Now rotate the plate. Open lid and streak again, following the diagram in the exercise book. Remember: you are picking up growth from quadrant one, and using this as your inoculum for quadrant two. 4. Flame loop; rotate plate, and repeat procedure for quadrants three and four. The proper wrist action and light touch takes practice. BSc(MolBiol) Lect 2.ppt

Sterilization and Waste disposal For MICR 2909 Lecture 2, 2001 Sterilization and Waste disposal Sterilization ensures killing/removal of ALL life forms Boiling kills most vegetative cells (Bacterial spores unaffected) Tyndallisation (c.1880): heat, 24hr, heat Dry heat (very high temperatures) Moist heat Autoclave: steam under pressure (121oC) Filtration (0.45 mm or 0.22 mm filters) Radiation (Gamma, UV, Ionizing) Other methods Sterilization and Waste Disposal Purpose To sterilize microbiological growth media both before and after inoculation with bacteria.   Principle One method of sterilization is the steam autoclave, which accomplishes the sterilization process by a combination of high temperature, pressure and time. This is a steam autoclave which operates much like a steam pressure cooker. Items to be sterilized are placed inside the autoclave chamber. To sterilize the contents, the autoclave must reach a temperature of 121oC for 15-20 minutes. In addition, the pressure must reach 15 lbs. per square inch. If any of these parameters--time, temperature and pressure--are not met, then sterilization may not be complete. All microbiological waste, including test tubes, plates, and any other contaminated materials must be autoclaved (sterilized) before disposal. BSc(MolBiol) Lect 2.ppt

Culture media formulation C & energy source (e.g., glucose) N source (organic or NH4+ or NO3¯ ) minerals (macronutrients, micronutrients) Macronutrients C, H, O, N, P, S - major K, Ca, Mg, Fe - minor (as cations) Micronutrients (trace elements) Mn, Zn, Co, Mo Ni, Cu (growth factors, vitamins) (agar)

Types of media General purpose Defined Allows growth of most bacteria, e.g., Nutrient agar Includes organic C, N, vitamins May have undefined components e.g., yeast extract, peptone Defined All components are pure compounds, not mixtures such as yeast extract E.g., glucose + (NH4)2SO4 + minerals for E. coli

For MICR 2909 Lecture 2, 2001 BSc(MolBiol) Lect 2.ppt Types of media Selective Favours one organism and limits growth of others Lacks some factor(s) E.g., fixed N, to select for N2-fixing bacteria Selective toxicity E.g., bile salts to select for Enterobacteriaceae Selective via incubation conditions E.g., gas composition (e.g., N2, 5% CO2, O2), temperature BSc(MolBiol) Lect 2.ppt

Types of media Differential Different bacteria/groups give different responses E.g., MacConkey agar: has lactose + peptone + indicator (neutral red) lactose fermenters  acid  pink colour non-lactose fermenters use peptone  neutral or alkaline  colourless

Enrichment Techniques For MICR 2909 Lecture 2, 2001 Enrichment Techniques Increase proportion of desired physiological class E.g., N2-fixers; cellulose-decomposers; photosynthetic bacteria Culture mixed population in selective medium and/or conditions E.g., fixed N-free; cellulose as sole carbon, energy source; anaerobic conditions in light, without organic C Sample treatment E.g., boil to kill vegetative cells, leaving spores BSc(MolBiol) Lect 2.ppt