The Compound Light Microscope

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MCB2010 Lab Notes ______

Ex1 (P1-4) and Ex2 (P5-12)

The compound light microscope Resolution = ability of the optics to discern 2 objects that are Close together Refractive index = the ability of material to bend light - light is bent as it pass from one material into another, e. g. air to glass. Immersion

 Oil has same reflective index as optical glass, prevent light Bending when used between glass slide and objective lens.

Working distance = the distance between the object and the objective lens when in focus the longer the length of the lens, the shorter the working distance Par focal lens = when one lens is in focus, the next lens in the series will also be approximately in focus Total magnification = power of ocular x power of objective lens

Tips in focusing 1. Lower mechanical stage. Insert slide on mechanical stage, move stage to make sure that slide moves freely without sticking. Move scanning lens in place. 2. While watching the stage from the side, bring stage all the way up. 3. While looking through the ocular, focus by turning the coarse Adjustment knob until you obtain a sharp focus. 4. With your eye still looking through the ocular, move the specimen to the middle of the vision field. 5. Switch to the next lens with a higher power. 6. Repeat step 3, 4 and then 5. 7. Repeat step 3, this time use the fine adjustment, then repeat step 4. 8. Rotate the nose plate to rest in-between the 40X and 100X lens 9. Add a drop of immersion oil, swing 100X lens in place. 10. Focus using the fine adjustment.

Focusing Select object to focus (usually colored objects) Focus Center object Change lens Focus, center object, change lens

Prepare microscope for storage Clean off oil from lens with lens tissue ONLY. Put scanning lens in place. Lower mechanical stage, center stage Coil up electric cord. Carry scope with both hands to storage. Replace plastic cover.

Answer Review questions 2 and 3 b, P12 of Lab Manual

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Ex 3(P13-28)

Slide to be observed in Lab: Bacterial types, 100X objective Identify the three common shapes of bacteria cells. Identify the arrangement of these cells.

Common shapes of bacteria Bacillus (bacilli), rod like cells Coccus (cocci), oval cells Spiral, curve cells Coccal-bacilli, short rods

Common arrangements Strepto- chains, e.g. Streptobacilli Staphlo- bunches, e.g. Staphlococci Diplo- in 2s, e.g. Diplococci

Photosynthetic Microbes – Includes Cyanobacteria and Algae

Slide to be observed: 1. Ocillatoria – up to 40X objective 2. Nostoc – 100X objective

Cyanobacteria

Originally thought to be more like algae, therefore, it is also known as Blue-green algae. Recently found to be more similar to bacteria than algae, hence renamed as Cyanobacteria (cyano=dark blue). Distinguishable from algae by being all prokaryotes and having only chlorophyll a. (chlorophyll = one of many photosynthetic pigments) But without chloroplast (chloroplast = membrane bound organelle containing chlorophyll).

Characteristics Prokaryotes, photosynthetic Unicellular or filamentous Contains only chlorophyll a No chloroplast Primary producers

Features that can be identified in your slides:

Akinetes = found in some filamentous cyanobacteria, often enlarged with thick walled,yellow or brown structures. Used as reproductive cells. Heterocysts = found in filamentous forms, thick walled, light green structures, used for nitrogen fixing.

Algae Slide to be observed: up to 40X 1. Volvox – Observe the nucleus and presence of organelles, oval cells forming colony 2. Spirogyra – observe the ribbon like chloroplast, nucleus, filamentous body.

Photosynthetic eukaryotes, possess chlorophyll a + one or more of chlorophyll b, c or d contained inside chloroplasts. Consists both non-motile or motile species. Algae are known as primary producers. (they form the beginning of food chains)

3 major groups of Algae 1. Non-motile, named according to pigments they contain: Green, Red and Brown algae. Observe the following slides in the lab:

Spirogyra - green algae

Volvox - green algae

2. Pigmented flagellates 3. Diatoms - possess rigid cell wall of silica

Summary of Characteristics of Algae

Non-motile algae pigmented flagellates Diatomes

Motility non-motile flagella gliding

Cell wall semi rigid thin or absent rigid with markings Contain silica

Eye spot absent present absent

Protozoa

Eukaryotes, include both free-living and parasitic forms, can be divided into 4 groups based on motility. One group, Sporozoa, is entirely parasitic and non-motile, a common example is the agent that causes malaria. Some parasitic protozoans involve a definitive host and an intermediate host, many are transmitted by insect vectors (carriers).

Protista - Protozoa 4 groups based on motility: 1. Sarcodina - Amoeba, move by psuedopods. 2. Mastigophora - Euglena, move by flagella. 3. Ciliophora - Paramecium, move by cilia 4. Sporozoa - All parasites, Plasmodium(Malaria parasite), none are motile.

Characteristics Unicellular Eukaryotes No cell walls Reproduce asexually Include free-living and parasitic forms Primary consumers

Slides of free living protozoa in lab Amoeba proteus huge in size compare to the parasitic amoebae, many Pseudopods can be identified, nucleus. Paramecium large shoe-shaped protozoa with cilia, well defined nucleus, mouth gullet and other vacuoles.

Eukaryotic, unicellular(YEAST) or colony forms(MOLD). The vegetative cell can be oval or filamentous.

MOLD Hypha = tubular form of vegetative fungal cell, usually joint end to end forming a long filament, can be septated (with cross-wall in between cells) or coenocytic (no cross- wall). Mycelium = a mass of many branched hyphae, usually visible by naked eye.

Many fungi have two morphological forms, a yeast form and a mycelial (MOLD)form, these Fungi are called dimorphic fungi.

Consult your text book concerning the asexual and sexual reproduction of fungi.

Slides in the lab: Rhizopus: spores are found in a swollen structure on top of a special stalk, the swollen sac is called sporangium and the spores are called sporangiospores. These spores are for asexual reproduction.

Penicillium: spores are not enclosed in the sporangium, they are not enclosed but are found on top of the sporangium. These spores are called conidiospores (meaning dust-like)

Saccharomyces: yeast, oval cells with vacuoles and granules, reproduced asexually thru budding.

Ex 4 Blood Typing (P29-34)

Blood typing is an example of the agglutination reaction, which Is a reaction between an antibody and an antigen.

Antigen and antibody reactions. Antigen - soluble or particulate, mostly proteins. Antibody-soluble, all proteins RBC = a particulate antigen Anti –A, anti-B, anti-D are all antibody containing reagents (sera) Blood typing - based on agglutination reaction between a particulate antigen (RBC) and the antibodies( anti-A, anti-B, anti Rh) specific for that antigen.

Blood type A has 'A' antigen on RBC - will be agglutinated by anti-A Blood type B has 'B'antigen on RBC - will be agglutinated by anti-B Blood type O has no 'A' or 'B' antigens on RBC - will not be agglutinated by anti-A or anti-B Rh + RBC has 'D' antigen on RBC - will be agglutinated by anti- D(anti-Rh)

Answer Critical Thinking questions 2 and 4, p 34

Preparation for Ex 5 (P35-40)

Ex 5 (P41-48)

Bacteria Isolation Instruments Inoculating loop ?for agar plates and agar slants, and broth tubes. Inoculating needle - for agar deeps. Cotton swab - for agar plates. Transfer pipettes - for broth tubes, agar pour plates. Spreader – spread agar plates.

Media Liquid - broth with nutrients to support growth Solid - broth supplemented with a solidifying agent, usually agar. Agar - extract from seaweed, no nutritious value. Liquid at 100C Solid at 40C and below

Forms of Media Agar plates - for growth and isolation(streak plate) Agar slants - for growth and stock culture Agar deeps - growth of bacteria in reduce oxygen conditions Broth tubes - growth in liquid media

Types of medium Simple or synthetic - chemically defined, all ingredients known, e.g. glucose salt broth. Complex - one or more of the ingredients are not defined chemically, e.g. nutrient extract containing medium

Ex 6 (P55-62)

Transfer techniques Loop sterilization - burning the wire loop or needle. Aseptic procedures: Flaming mouth of glass containers ?to create an Updraft to prevent aerial contamination.

To prevent contamination: Always keep cover or cap on, open only when Needed, close ASAP when finish.

Culture techniques Put all culture in class tray. Tubes should be upright. Agar culture plates should be placed lid side facing down. Everything needs to be labeled. Move or remove things carefully.

Growth characteristics in broth Sediment - microbes at bottom of tube. Flocculent - clumps of microbes in suspension. Pellicle - microbes on top, forming a membrane. Turbidity - microbes form a cloudy suspension.

Aseptic Transfer A) Flame loop to red hot, cool for 1 min. B) Remove cap, flame month of tube C) Pick up culture, flame month of tube, replace cap. D) repeat (B) for receiving tube. E) Deposit culture from (C) to receiving tube. F) Flame tube and recap, flame loop.

Answer Review questions 4 - 9; Critical thinking question 3 p 60-61

Ex 7 (P63-70)

Streaking for isolation Mark four quadrants on the underside of a plate. Flame loop, cool, pick up culture. Deposit culture in sector one, flame loop, cool, streak on sector 1, flame loop, cool. Repeat above for sector 2. Repeat for sectors 3 and 4 with no loop flaming.

Ex 8 (P71-74)

Bacterial Movement Brownian movement: - Cell being moved by the vibration of water molecule surrounding cell, no consistent directions. Movement by flagella - Cell propelled by circular movement of flagella, distinct direction followed by tumbling and change of direction.

Answer Review questions 1 and 3 p73 -74 on Lab manual

Preparation for Ex9 (P75-77)

Ex 9 (P79-84) Bacterial Stain Stain is used to increase the contrast between cells and background.

Two chemical groups in a stain Chromophore - imparts color to the stain. Auxochrome - imparts chemical properties such as electrical charges to stain. Bacterial Stains Basic stains - carry a positive charge, usual stains for bacterial cells, which carry negative charges on cell wall. Acidic stains - carry a negative charge, generally used for background staining.

Smear Preparation 1. Spread or create a suspension of bacterial cells on a clean glass slide(dime size). 2. Air dry thoroughly. Ideal thickness ?ability to see printed words through the dried smear. 3. Heat fix by passing several times on top of flame.

Simple Stain – using one stain only Cover smear with stain for 1 min. Tilt to drain stain from slide. Rinse thoroughly with water. Blot dry and examine.

Answer review questions 3 ans 4 P84

Ex 10 (P85-90)

Gram Stain Primary Stain - Stain with Crystal Violet for 1 min. Tilt to remove stain, rinse with water. Mordant - Add Gram’s iodine, stain for 1 min. Tilt to remove solution, rinse thoroughly. Decolorization - Tilt slide, drip Ethyl alcohol over smear to decolorize smear. Observe the lower edge of the smear carefully, stop alcohol decolonization as soon as the run off from the smear is clear. Rinse with water, shake off excess. Counter Stain - Add safranin, stain for 50 seconds. Tilt to remove stain, rinse thoroughly. Blot dry.

Answer All revuew questions p89-90

Ex 11 (P91-96)

Acid-fast stain: A differential stain which distinguish acid fast organisms from Non-acid-fast organisms, based on the presence of Mycolic acid, a waxy substance in the cell wall of acid-fast organisms. The primary stain is driven through the cell wall by heat. An example of acid-fast organism is:

Mycobacterium sps.

Ziehl-Neelsen acid-fast stain includes the following steps: 1. Preparation of bacterial smear 2. Air drying of smear 3. Heat fixing of smear 4. Primary stain - Carbo-fushin 5. water rinse 6. Decolourisation - acid alcohol 7. water rinse 8. counter stain - Mythylene blue 9. water rinse and blot dry

Answer Review questions 2, 3, Critical Thinking 1 and 3

Ex 12A (P97-98)

Endospore stain: A special stain which stains endospores, commonly present in species of Bacillus and Clostridium.

Steps in performing the Schaeffer-Fulton Endospore stain 1. Prepare bacterial smear 2. Air dry smear 3. Heat fix smear 4. Primary stain - Malachite green with heat on a steam bath 5. Water rinse and decolonization 6. Counter stain with Safranin O 7. water rinse and blot dry

Ex 12B (98-103)

Capsule stain: A special stain which reveal the polysaccharide capsule of some bacteria. Capsular material is soluble in water, copper sulfate is used to wash bacteria smear isnstead.

Steps in performing the capsule stain (direct staining using basic Stain)

*DO NOT PREPARE SMEAR THE REGULAR WAY; NO HEAT FIXING IS NECESSARY

1. Add 1 drops of crystal violet on a clean slide. 2. Aseptically add loopful of culture and mix with the crystal violet. 3. Prepare a thin film smear as directed. 4. Air dry and examine with oil immersion lens.

Answer review questions 1, 2, 3b, 3c, 4, 5 on page 102-103

Ex 13 (P105-108)

Negative Staining: Staining the background instead of the bacterial cells by use of an acidic stain, cells and associated structures such as capsule will appear as empty objects on a color background.

Steps in performing a negative stain *DO NOT PREPARE SMEAR THE REGULAR WAY; NO HEAT FIXING IS NECESSARY 1. Add a drop of Nigrosin to one end of a clean slide. 2. Aseptically transfer a loopful of bacteria and mix well with stain. 3. Prepare and thin film smear as directed. 4. Air dry and examine with oil immersion lens.

Answer all review questions Introduction to Ex 15 (p113-p115)

Ex 15 Enumeration of bacteria growth curve (p117 – 118)

Bacterial growth curve typically, consists of 4 phases: 1. Lag phase - no increase in cell number, bacterial is preparing for cell division by duplication of DNA and production of various materials needed for cell multiplication. 2. Log growth phase - more cells are being born than die, resulting in greatest increase in cell number per time interval. 3. Stationary phase - nutrients are getting used up by the rapidly dividing cells, resulting in equal number between new cells generated and those that died. 4. Log death phase - continue depletion of nutrients, more cells are dying than being born.

Exercise modifications: Procedure 1. Obtain 6 tubes of BHI broth, label as 0, 30, 60, 90, 120, 150 min. 2. To each of the above labeled tube, add 0.1 or 2 drops of E. coli stock culture, mix well. 3. Put all tubes except the 0 min in 37 C incubator. 4. Transfer the content of the 0 min tube to a measuring tube. Measure and record the Optical Density of the content in tube 0. 5. Transfer content back to the culture tube, discard both tubes. 6. At the end of each time interval as marked on each tube, repeat Step 4 and 5 above. 7. Plot graph using the data obtained.

Spectrophotometer count

Light Transmission in spectrophotometer reading - Bacterial concentration is indirectly proportional to amount of light transmitted.

Optical density in spectrophotometer reading - Bacterial concentration is directly proportional to amount of light absorbed.

Requirements before reading sample: Blank (zero) reading necessary with the same medium as the culture to be measured. Standard curve showing relationships between absolute bacterial number an spectrophotometer readings had to be constructed.

Answer review questions 1 & 4 on page 122

Introduction to Ex 16, 17 (p127-p129) Ex 16 A(p131 – 134)

Antimicrobial agents

Antimicrobial agents includes: a) antibiotics, which are produced by living organisms, usually microorganisms, to inhibit growth of others. b) antiseptics and disinfectants, which are often chemical compounds made by man to inhibit growth of microbes.

Test for susceptibility of micro-organisms towards any agents involve growing Of micro-organisms in the presence of the substance to be tested. The one that you are going to do in the lab is the disc diffusion test where antimicrobial agent in a paper disc is allowed to diffuse into the medium which had been previously seeded with a lawn of bacteria for the test. The size of the area where there is no bacterial growth indicated the zone of inhibition. The zone of inhibition is measured in mm and represents the diameter of the clear circle where there is no growth of bacteria.

The Kirby-Bauer antibiotic sensitivity test depends on the ability of antibiotics diffusion into surrounding medium. A standardised plate, Mueller-Hinton plate, is used to limit various physical factors that may affect the diffusion rate.

MIC = minimum inhibitory concentration, which is the minimum concentration of antimicrobial agent needed to inhibit the growth of a bacterium. The MIC is different for each bacteria and varies with different antimicrobial agent. The effectiveness of your laboratory test should be compared to a standard chart to see if it falls within the effective range.

Answer review questions 3 page 148

Ex 17 B (141-146)

Exercise Modification: Material: 5 mm filter paper discs Solutions of various disinfectants and antiseptics Procedure: 1. Obtain a Mueller-Hinton agar plate, mark off the appropriate number of sectors, one for each solution to be tested, label. 2. Using forceps, Dip and saturate a paper disc with disinfectant solution, drain excess solution by touching the side of the container. 3. Carefully lay down the disc onto the middle of the labeled Sector, tap disc gently to ensure contact with agar. 4. Repeat the above until you finish with all the solutions to be tested, secure lid and incubate till next lab.

Temperature as a means of control of microbial growth. Refer to your lab manual, P149, Growth Temperatures

Bacteria can be separated into several group based on their growth Temperature range:

Psychrophiles 0 - 200 C Mesophiles 25 - 400 C Thermophiles 45 - 650 C

Types of Heat for Control of microbial growth: Dry Heat- not efficient, requires long exposures, for glass wares and solids.

Moist Heat- efficient, various types: Autoclave - steam under pressure, for sterilization, 15 PSI, 1210C, 15 minutes. Boiling - not effective for endospores. Pasteurization - prevent spoilage, 630C for 30 minutes.

Answer questions 2, 5, 6 p 152.

See lab manual p155 for theory

Answer questions 1, 2 and Critical thinking 1, 2 p160

Radiation Control of Microbial Growth, two major forms of radiation that can affect the growth of micro-organisms: Ionizing Radiation - High energy, high penetration, generates free electrons and free radicals, damage DNA molecules. Example: X rays, Gamma rays.

Non-ionizing Radiation - Low energy, low penetration. Example: UV light, generates thymine dimers, generates mutations in DNA, reversible through repair mechanisms which operate in darkness.

See lab manual p169 for principle

Direct Microscopic Count Hemocytometer - fixed volume counting chamber, count 5 squares in center grid. Calculation average of 5 squares x 25 x DF DF= dilution factor

Water Analysis Fecal contamination of water Indicator: E. coli 3 Basic tests presumptive - lactose fermentation with gas production confirmed - growth of organism on selective and differential media (EMB agar) completed - gram stain and growth on lactose broth with acid and gas production

Answer Review Questions 1, 4 page 182

Sugar Fermentation Uses glycolysis to generate energy – Glucose Broken down to form Pyruvic acid. Pyruvic acid is the intermediate, it is further broken down either in: Alcohol fermentation - end product is alcohol and gaseous CO2 Homolactic fermentation - end product is lactic acid Heterolactic fermentation - end products are mix acids, alcohol, gas

Sugar Fermentation Broth Tube Contents: Nutrient Broth - supports proteolysis(peptonization)for non- fermenters, produce basic end product. Sugar - fermentation produces acids with or without gas, acid decreases pH. Phenol red - acid base indicator, yellow = acid red = base Durham tube - inverted tube for gas collection.

Answer review questions 1,4, 5 page 191 -192

24A Microbial Exoenzymes

Carbohydrate Hydrolysis Starch = complex carbohydrate Building blocks = simple sugar (glucose) Hydrolytic Enzyme Amylase breaks down starch to glucose. Test for enzyme activity = presence or absence of starch. Iodine + starch = Blue complex

Microbial Exoenzymes

Gelatin Hydrolysis Building blocks = amino acids. Hydrolytic Enzyme gelatinase breaks down gelatin to amino acids. Test for enzyme activity - lose of ability of gelatin to gel at room or temperature.

24B Microbial Exoenzymes

Lipid Hydrolysis Building blocks = glycerol (an alcohol) and fatty acids. Hydrolytic Enzyme lipase breaks down lipid to fatty acids and glycerol. Test for enzyme activity(lipolysis) presence of lipid = opaque agar absence of lipid = clear agar.

Microbial Exoenzymes

Casein(milk protein) Hydrolysis Building blocks = amino acids. Protease Enzyme breaks down casein to amino acids. Test for enzyme activity - proteolysis presence of proteolysis = clear zone absence of proteolysis = opaque agar

Nitrate Reduction

Nitrate reagent A + B NO3 ------NO2 Red

Nitrate reagent A + B NO3 ------NO2 ------NH3 ------N2 colorless

Nitrate reagent A + B NO3 ------colorless

Nitrate reagent A + B NO3 ------NO2 Red Manual reduction using zinc

Answer review questions 3, 4 page 202

IMViC Test I = Indole production from oxidation of amino acid Tryptophan

SIM agar deeps 1 semi-solid agar for motility motile bacteria – growth throughout agar non-motile bacteria – growth along stab 2 Tryptophan for Indole production Presence of Indole Turns Kovacs reagent red 3 Test for Hydrogen Sulfide production Black precipitate of Ferric sulfide in agar

Methyl Red Test Test for the product of Glucose oxidation Stable acid – turns MR reagent to red Example – E. coli Acetoin – non acidic, turns MR reagent yellow. Example – E. aerogenes

VP Test Voges Proskkauer test Test Product of glucose oxidation Neutral products – turns Barritt’s reagent pink. Example: E. aerogenes

Citrate Test Test for the fermentation of citrate as a sole source of carbon. Positive results: 1. Growth and/or 2. Alkaline product turns Bromthymol Blue from green to blue.

Catalase Test Results of survival in oxygen Toxic products - H2O2 and superoxide Enzymes needed to detoxification Catalase – converts H2O2 to water and oxygen. Superoxide dismutase – converts superoxides to H2O2.

Use of Selective or Differential Media

Selective and Differential Media Mannitol Salt agar Salt selects for halophile Mannitol differentiates Mannitol fermenting from non-fermenting

organisms.

EMB agar dyes inhibits Gm +, selects for Gm - Lactose differentiate fermentation + vs – ve organisms. Also Differentiate heavy vs light acid producers.

MacConkey agar – very similar to EMB Crystal violet dye inhibits Gm +, selects for Gm -.

Lactose differentiates fermenting from non-fermenting organisms.

Differential Media Blood Agar – test for hemolysis of RBC Alpha – incomplete hemolysis, release and partial degradation of hemoglobin, red or green zone. Beta – complete hemolysis, total degradation of released hemoglobin, clear zone. Gamma – no hemolysis, absences of any zones.

Selective Media Phenylethyl Alcohol Agar Select for Gm +, inhibits Gm - organisms. Generally used for isolation of Staphylococcus and Streptococcus from mixture.

Rapid Identification Test Enterotube Commercially produced rapid test for the identification of Enterobacteriaceae Advantages – fast results, less labor intensive, provides indications for early treatments.

Answer questions

p 212 Question 6; p. 216 #2 and 3; p. 222 # 1 and 2

Good Luck, you have make it this far. 2 more hurdles to get thru and you will finish.