BIO2010 MICROBIOLOGY LAB PRACTICAL #1 REVIEW 1. Brightfield Microscope:

a. Know the parts of the microscope and the function of each partb. Know the 3 lens systems: ocular, objective, and condenserc. Know the total magnification calculations with the 3 lens in placed. Relationship of magnification with:

i. Field of view – inversely proportional (decreases with increase in magnificationii. Working distance – decreases with increase in magnificationiii. Depth of field – decreases with increase in magnificationiv. Resolution – increases with increase in magnification

e. Immersion oil – function is to decrease refraction (bending) of light rays; when oil is used , the aberrations are diminished and the resolution is enhanced

f. Parfocal – if a specimen is in focus at one power of magnification, it should be approximately in focus at all other powers. You can get a specimen in focus at 10X and then use only the fine adjustment knob to bring it clearly into focus at a higher power of magnification.

g. Know how to store and handle the microscope properly2. Know about other types of microscopes; their use, advantages and disadvantages3. Wet Mount:

a. This procedure is used to determine true directional motilityb. 400X is the highest total magnification used for viewing; oil immersion objective

(100X) cannot be usedc. Alternative wet mounts – hanging drip, motility agar

4. Culture Media:a. Know the differences between pure, mixed, and contaminated biological culturesb. Culture temperatures that are used in laboratory experiments – 250C (ambient room

temperature; 300C, 370C; DO NOT FORGET UNITSc. Basic medium used in the lab – Nutrient Broth provides the necessary carbon,

nitrogen, vitamin sources for most MO’s that we will be studying.d. Fastidious MO’s require the use of enriched culture media; trypticase soy agar (TSA)

and blood agar (BAP) are commonly used in laboratory experiments.e. Solid media – this media is made by adding 1.5% agar, a polysaccharide from

marine (red) algaei. Semi-solid media – add 0.5% agarii. Forms of agar culture media: Petri plate, slant, deep tube

f. Selective media – this media contains chemical to suppress undesired MO’s from growing; i.e. salt is media is selective for Staphylococcus sp. Or bile salts in media to select for enteric gram negative MO’s.

g. Differential media – this media is designed to distinguish between different species of MO’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 taken place.

5. Ubiquitous: omnipresence; existence everywhere at the same time6. Handwashing:

a. Two populations of MO’s are present on the skini. Transient – easily acquired and discardedii. Residential – deeply embedded in the layers of the epidermis

1

b. Calculating numbers of bacterai. If 0.2 ml is added to a Petri plate and agar added; Following incubation there

are 35 colonies on the plate, then the following calculations must be used.1. We know that the 35 CFU on the plate were put there in the 0.2 ml

sample. So there are 35 CFU per 0.2 ml, but we want our answer in CFU per 1 ml, so we must multiply by a correction factor of 5/5

2. 35 CFU x 5 = 175 CFU = 1.75 x 102 CFU/ml 0.2 ml 5 1 ml

ii. Other correction factors:1. Multiply by 10/10 for CFU/0.1 ml2. Multiply by 2.5/2.5 for CFU/0.4 ml

c. The countable number of colonies must be between 30 and 300 CFU per plate7. Pour Plate:

a. Liquefaction of agar (melting temperature) – 1000Cb. Holding temperature – 500Cc. Pouring temperature – 450Cd. Resolifidication temperature – 400Ce. If agar is too cold when pouring, lumps will occur in the plate or the agar will not pourf. If the agar is too hot when pouring, condensation will occur in the plate plus many of

the MO’s will apt to be killedg. Always incubate bacterial culture plates “upside” down

8. Aseptic ‘”Transfer:a. Asepsis – without contaminationb. Know how to perform the technique, including instruments used (inoculating needle

or loop)c. Know possible areas or points where contamination can take place

9. Streaking for Isolation:a. Know the process involved in the techniqueb. Be able to recognize a good plate from an inadequate plate (poor isolation – no

colonies in the 3rd quadrant)10.Bacterial Smear:

a. Know the appropriate concentration needed to make an excellent 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 MO’s - ~ the size of a quarter on the slide)

b. Air dry ~ 15 minutesc. Heat fix – this will kill the MO’s and induce adherence to the slide

11.Simple stain:a. Procedure – Methylene Blue solution for 1.0 minute; then rinse with tap waterb. This will demonstrate only shape and arrangement of MO’s

12.Gram stain (know this procedure in detail)a. Be able to write all steps involved in Gram staining the MO’sb. Gram positive – purple in colorc. Gram negative – red or pink in colord. Differential stain – know the steps involved with this type of staining

2

e. Gram stain reaction for the following MO’si. Bacillus – gram pos rod, may be in chains; old cultures gram variableii. Streptococcus – gram pos cocci, chains of varying lengthsiii. Staphylococcus – gram pos cocci, grape-like clustersiv. Rhodospirillum – gram neg spirillumv. Escherichia – gram neg coccobacillusvi. Neisseria – gram neg diplococcicvii. Serratia – gram neg bacilliviii. Mycobacterium – gram variable bacilli

13.Spore stain:a. Primary stain – malachite green solutionb. Counterstain – safraninc. Genera that form endospores

i. Bacillusii. Clostridium

d. Recognize the red and green colors found on a smear14.Ziehl-Neelsen Acid Fast stain

a. Primary stain – carbol fuschsin solutionb. Counterstain – Loeffler’s methylene blue solutionc. Genera that of acid fast

i. Mycobacterium – tuberculosis, leprosyii. Nocardia – nocardiosis

d. Acid Fast – pink color due to mycolic acide. Non-acid fast – blue

15.Bacterial structures:a. Be able to recognize inclusion bodies, and the various types of flagella

i. Monotrichous (polar) single flagella; Pseudomonas aeruginosaii. Amphitrichous (either end of cell): Spirillium voluntansiii. Lophotrichous (tufts of flagella on one end of cell): Helicobacter pyloriiv. Peritrichous (flagella all over the cell): Proteus vulgaris

b. Know the purpose of the structurec. Know the purpose of inclusion bodies

16.Know the various types of laboratory equipment used in microbiology17.Be able to identify the following MO’s, the disease associated with the MO’s, the Kingdom

to which each MO’s belongsa. Aspergillus nigerb. Penicillum notatumc. Rhizopus nigricansd. Trichinella spiralise. Trypanosomaf. Mixed diatomsg. Saccharomyces cerevisiaeh. Bacterial types (as you observed from the various slides)

18. Cultural Characteristics of Bacteriaa. On agar plates – colony shape, margin, size, elevation, pigmentation (color, diffusible

or non-diffusible), ecophenotypic variation.b. On agar slants – even, irregular or spreading growth.c. In broths – sediment, turbid, pellicle

3