BIO2010 MICROBIOLOGY

1. Brightfield Microscope:

LAB PRACTICAL #1 REVIEW

a. Know the parts of the microscope and the function of each part b. Know the 3 lens systems: ocular, objective, and condenser c. Know the total magnification calculations with the 3 lens in place d. elationship of magnification with: i. !ield of view " inversely proportional #decreases with increase in magnification ii. $or%ing distance " decreases with increase in magnification iii. &epth of field " decreases with increase in magnification iv. esolution " increases with increase in magnification

e. 'mmersion oil " function is to decrease refraction #bending( of light rays) when oil is used , the aberrations are diminished and the resolution is enhanced f. *arfocal " if a specimen is in focus at one power of magnification, it should be appro+imately in focus at all other powers. ,ou can get a specimen in focus at 1-. and then use only the fine adjustment %nob to bring it clearly into focus at a higher power of magnification. g. Know how to store and handle the microscope properly /. Know about other types of microscopes) their use, advantages and disadvantages 3. $et Mount: a. 0his procedure is used to determine true directional motility b. 1--. is the highest total magnification used for viewing) oil immersion objective #1--.( cannot be used c. 2lternative wet mounts " hanging drip, motility agar 1. 3ulture Media: a. Know the differences between pure, mi+ed, and contaminated biological cultures b. 3ulture temperatures that are used in laboratory e+periments " /4 -3 #ambient room temperature) 3--3, 35-3) &6 760 !6 890 :7'0; c. Basic medium used in the lab " 7utrient Broth provides the necessary carbon, nitrogen, vitamin sources for most M6<s that we will be studying. d. !astidious M6<s re=uire the use of enriched culture media) trypticase soy agar #0;2( and blood agar #B2*( are commonly used in laboratory e+periments.

e. ;olid media " this media is made by adding 1.4> agar, a polysaccharide from marine #red( algae i. ;emi?solid media " add -.4> agar ii. !orms of agar culture media: *etri plate, slant, deep tube f. ;elective media " this media contains chemical to suppress undesired M6<s from growing) i.e. salt is media is selective for Staphylococcus sp. 6r bile salts in media to select for enteric gram negative M6<s. g. &ifferential media " this media is designed to distinguish between different species of M6<s) i.e. lactose fermentation) there is usually an indicator added to the media #neutral red or phenol red( to yield a color change #red to yellow( to indicate that fermentation of lactose has ta%en place. 4. :bi=uitous: omnipresence) e+istence everywhere at the same time @. Aandwashing: a. 0wo populations of M6<s are present on the s%in i. 0ransient " easily ac=uired and discarded ii. esidential " deeply embedded in the layers of the epidermis

b. 3alculating numbers of bactera i. 'f -./ ml is added to a *etri plate and agar added) !ollowing incubation there are 34 colonies on the plate, then the following calculations must be used. 1. $e %now that the 34 3!: on the plate were put there in the -./ ml sample. ;o there are 34 3!: per -./ ml, but we want our answer in 3!: per 1 ml, so we must multiply by a correction factor of 4B4 /. 34 3!: + -./ ml 4 C 154 3!: C 1.54 + 1-/ 3!:Bml 4 1 ml

ii. 6ther correction factors: 1. Multiply by 1-B1- for 3!:B-.1 ml /. Multiply by /.4B/.4 for 3!:B-.1 ml c. 0he countable number of colonies must be between 3- and 3-- 3!: per plate 5. *our *late: a. Di=uefaction of agar #melting temperature( " 1-- -3 b. Aolding temperature " 4--3
2

c. *ouring temperature " 14-3 d. esolifidication temperature " 1--3

e. 'f agar is too cold when pouring, lumps will occur in the plate or the agar will not pour f. 'f the agar is too hot when pouring, condensation will occur in the plate plus many of the M6<s will apt to be %illed g. 2lways incubate bacterial culture plates EupsideF down G. 2septic HF0ransfer: a. 2sepsis " without contamination b. Know how to perform the techni=ue, including instruments used #inoculating needle or loop( c. Know possible areas or points where contamination can ta%e place I. ;trea%ing for 'solation: a. Know the process involved in the techni=ue b. Be able to recogniJe a good plate from an inade=uate plate #poor isolation " no colonies in the 3rd =uadrant( 1-. Bacterial ;mear: a. Know the appropriate concentration needed to ma%e an e+cellent smear #use a loop of water with a small amount of the specimen from the agar slant or plate) rotate the loop gently several time in order to have a monolayer of M6<s ? K the siJe of a =uarter on the slide( b. 2ir dry K 14 minutes c. Aeat fi+ " this will %ill the M6<s and induce adherence to the slide 11. ;imple stain: a. *rocedure " Methylene Blue solution for 1.- minute) then rinse with tap water b. 0his will demonstrate only shape and arrangement of M6<s 1/. 8ram stain #know this proc !"r in ! t#i$( a. Be able to write all steps involved in 8ram staining the M6<s b. 8ram positive " purple in color c. 8ram negative " red or pin% in color d. &ifferential stain " %now the steps involved with this type of staining

e. 8ram stain reaction for the following M6<s i. Bacillus " gram pos rod, may be in chains) old cultures gram variable ii. Streptococcus " gram pos cocci, chains of varying lengths iii. Staphylococcus " gram pos cocci, grape?li%e clusters iv. Rhodospirillum " gram neg spirillum v. Escherichia " gram neg coccobacillus vi. Neisseria " gram neg diplococcic vii. Serratia " gram neg bacilli viii. Mycobacterium " gram variable bacilli 13. ;pore stain: a. *rimary stain " malachite green solution b. 3ounterstain " safranin c. 8enera that form endospores i. Bacillus ii. Clostridium d. ecogniJe the red and green colors found on a smear

11. Liehl?7eelsen 2cid !ast stain a. *rimary stain " carbol fuschsin solution b. 3ounterstain " Doeffler<s methylene blue solution c. 8enera that of acid fast i. Mycobacterium " tuberculosis, leprosy ii. Nocardia " nocardiosis d. 2cid !ast " pin% color due to mycolic acid e. 7on?acid fast " blue 14. Bacterial structures: a. Be able to recogniJe inclusion bodies, and the various types of flagella i. Monotrichous #polar( single flagella) Pseudomonas aeruginosa ii. 2mphitrichous #either end of cell(: Spirillium voluntans
4

iii. Dophotrichous #tufts of flagella on one end of cell(: Helicobacter pylori iv. *eritrichous #flagella all over the cell(: Proteus vulgaris b. Know the purpose of the structure c. Know the purpose of inclusion bodies 1@. Know the various types of laboratory e=uipment used in microbiology 15. Be able to identify the following M6<s, the disease associated with the M6<s, the Kingdom to which each M6<s belongs a. Aspergillus niger b. Penicillum notatum c. Rhi opus nigricans d. !richinella spiralis e. !rypanosoma ". Mi#ed diatoms g. Saccharomyces cerevisiae h. Bacterial types $as you observed "rom the various slides%
18. 3ultural 3haracteristics of Bacteria

a. 6n agar plates " colony shape, margin, siJe, elevation, pigmentation #color, diffusible or non?diffusible(, ecophenotypic variation. b. 6n agar slants " even, irregular or spreading growth. c. 'n broths " sediment, turbid, pellicle