Microbiology Lab-1

compound microscope (2 lens between eye and object)

brightfield compound microscope

• scanning (4x)
• low power (10x)
• high dry (40x)
• oil immersion (100x)

multiply magnification of ocular (10x) by magnification of objective lens.

oil immersion

ability of lens to reveal fine detail or two point distincly separated

greatest when 2 objects are seen as distinct even though they are close together. the smaller the distance the better resolving power.

refracted when emerged from slide b/c of change in density as light passes from glass to air

light rays continue without refraction b/c immersion oil has same refractive index ax glass. as light rays pass through lens they are bent to converge at focal point where image is formed.

periphery may be fuzzy when you bring center of microscope field into focus d/t curvature of lens which results in multiple focal points.

• carry microscope with both hands
• observe slide with both eyes open
• focus slowly/carefully
• adjust light. more needed with higher resolution
• focus under low power first
• focus slide under 40x before going to oil immersion
• keep stage clean
• use only lens paper for cleaning

remagnifies image formed by objective lens. (eyepeice)

transmits image from objective lens to ocular lens

primary lens to magnify specimen

holds microscope slide in position

focus light through specimen

controls amount of light entering condensor

light source

make sure numeric measurement on each lens is same as iris diaphragm (understage)

particles and microorganisms all vibrate at same rate and maintain relative positions d/t molecules in liquid striking an object, causing it to shake or bounce.

motile organisms move from one place to another in a directed manner, sometimes as if to spin and roll.

• stir contents of container
• using pipette transfer small drop to slide
• lay coverslip over drop
• place onto sgtage and observe with low power (10x)
• observe slide under high power lens (40x). bacteria should now be visible

slow down microbes so easily seen

Anton Van Leeuwenhoek in 1600 described rod, coccus, spiral

• diplococci- pairs
• streptococci- row of 4+
• staphylococci- irregular clumps resemble grapes

aka bacillus , occassionaly found in diplobacilli and streptobacilli but not cocci.

• spirochetes- flexible, waving forms with several coils
• spirilla- rigid with one or several curves. if they are short without complete coils then called vibrios.

variations in shape and size of bacteria

• read procedure prior
• organize lab space
• dont rush
• use test tube rack
• hold loop secure in dominant hand
• dont hold test tube by cap
• sterilize loop using bunsen burner until red at 45 degree
• pass tops of tube through flame 1-2 x before recap
• incubate petri dish agar side up and lid side down
• hold all lids/caps in hand not on table
• clean space before and after lab

• extract from marine red algae
• remains solid during microbial growth
• few microbes degrade it
• liquifies at 95-100 celcius remains liquid until cool at 45 celcius

provide lare surface area for exam of colonies.

visible growth on agar surface that started out as 1 single bacterium and replicated self many times.

orginal bacteria that turn into colony during incubation

• first pass with culture sample
• second pass with sterile loop
• third pass with sterile loop
• leave space between first and third pass

colony not touching upon any other colony

streak plate used as dilution technique.

dilute out organism in mixed culute until cells are seperated from each other enough that single bactyeria become isolated in discrete location on agar surface. plate then incubated and cells undergo division resultint in visible growth upon surface of agar.

colored molecules positively charged and attracted to negative charged bacterial cell. this results in cell becomeing colored against a white background.

• methylene blue
• crystal violet
• safranin (red)

kills bacteria adn ensures that smear will stay attached to slide. it distorts specimen to small extent so dont overheat.

• use loop, place loopful sterile water on slide
• use same loop, transfer small amt bacteria from single colony into water
• let dry completely (or will wash off)
• heat fix slide (pass thru flame 2-3 x)
• add few drop methylene blue/crystal violet/safranin to slide
• leave for 1 min
• use deionize water to rinse off slide
• coverslip to slide
• observe on 10x then 40x

• stain background NOT bacteria
• bacteria appears clear against dark background

ionic repulsion- bacteria and acid stain both have negative charge

when other stain techniques dont show cell size/morphology.

bacteria less distorted so visualize easier

• small drop of nigrosin at end of slide (dont spread let dry)
• flame loop and add small loop of culture onto drop of nigrosin
• reflame loop
• using edge of another slide, spread drop to produce smear varying from dark at one end to gray
• let smear air dry - DO NOT HEAT FIX

identify and classify bacteria

differntial stain allowing classification of bacteria as either positive or negative

1884 Hans Christian Gram discovered when he attempted to stain cells and found some lost color when excess stain was washed off

• pick up loopful of sample using sterile loop
• smear sample all around slide and let air dry (dont blow on it)
• flame loop again
• heat fix slide
• apply primary stain to stain all bacteria purple (crystal violet- alkaline dye) 60 sec
• wash off with deionize water
• apply mordant (iodine) 60 seconds
• wash off with deionize water
• apply decolorizing agent (alcohol) only small amt until no large amt of purple wash out
• IMMEDIATE wash with deionize water
• apply secondary stain/counterstain (safranin) 60 sec
• wash with deionize water and blot dry

crystal violet - stains all bacteria purple (alkaline dye)

mordant- iodine (combines with crystal violet in cell to from crystal-iodine complex)

decolorizing agent- alcohol (primary stain washed out/decolorized of some bacteria while others remain purple)

secondary stain/counter stain- safranin (alkaline dye stains decolorized bacteria red)

• red= gram negative
• purple= gram positive

• chemical and physical properties in cell walls.
• crystal violet picked up by all bacteria, iodine react with crystal violet in cytoplasm to form CV-1
• gram negative= decolorize agent dissolves outer lipopolysaccharide layer and CV-1 washes out through thin layer of peptidoglycan

cool loop for 30 sec so bacteria pick up wont be killed. if too hot will splatter medium and move bacteria into air

• differential stain discovered by Paul Ehrlich 1882
• TB retain primary stain even after washing with acid alcohol.
• used as diagnostic tool for mycobacterium /nocardia as they contain pathogenic species

• most bacteria decolorize by alcohol.
• acid fast: mycobactrium, nocardia, goronia, tsukamurella

result of relative solubility of carolfuchsin and impermeability of cell wall.

fuchsin more soluble in carbolic acid (phenol) then in water and carbolic acid becomes soluble easier in lipid then alcohol. so carbolfuchsin has higher affinity for lipid then alcohol and will remain with cell wall when washed with alcohol.

• heat fix a smear
• cover smear with carbolfuchsin (leave for 5 min)
• wash slide with deionize water
• without drying, wash smear with alcohol until no red color runs off slide
• wash smear with deionize water
• counterstain with brilliant green(1 min)
• wash with deionize water and blot dry

clumpy

negative stain that stains negative space around bacteria for visual of capsule or slime layer

• negative charged congo red chemical stains negative space
• alkaline Manevals reagent chemical stain bacterial cells

klebsiella pneumoniae- mild pathogenic

• small drop of congo red at end of slide- let air dry/and dont spread
• flame loop and add small amt of culture to drop of congo red
• reflame loop
• using end of another slide, spread drop to produce smear
• let air dry- DO NOT HEAT FIX
• after dry, cover with maneval reagent (1 min)
• wash with deionize water and blot dry

stress resistant form of bacterium allows for survival in poor environment conditions

resistant to heat and chemical d/t production of outer layer of tough keratin

steam to stress bacteria into forming endospores

visible as small green structure

• central- middle of cell
• terminal- end of cell
• subterminal- between middle and end of cell

• spherical
• elliptical

• prepare heat fix smear
• put paper towel on top of smear and place over steming beaker under fume hood
• soak paper towel with malachite green (leave 7-10 min)
• paper towel must remain soaked and add more green prn
• remove slide, place paper towel in trash
• rinse with deionize water
• counterstain with safranin (1 min)
• rinse with deionize water and blot dry

when nutrients deplete, gram positive bacteria form these resting cells internal to bacterial cell membrane

• formation of endospore:
• spore septum isolate new replicated DNA and small part of cytoplasm
• plasma membrane surround DNA
• spore septum surround isolated portion to form forespore
• peptidoglycan layer form between membranes
• spore coat forms
• endospore is freed from cell