Download Exercise_2

2A: Microscopy
 Post Lab 2 is assigned today and due by the
time your lab meets next.
 Pre Lab 3 will be available on Wednesday at 5
PM and is also due by the time your lab meets
next.
 LNA Bacteria is assigned today, and due by the
time your lab meets next*.
 Pre-Lab Write Up for LNA 3 is due within the
first 5 minutes of lab next week.
*You will have time at the beginning of week 3 to look at your bacterial
plates and complete your LNA
 Develop working knowledge of a brightfield light
microscope
 Discern between different types of microscopy
 Practice techniques: objectives, oil, wet mount,
measurements
 Practice with the objectives, focusing, and positioning
using the prepared slides.
 Use your lab manual as a reference if you are having
trouble pages 30-31.
Bacterial Observation
.
 Sphere
 cocci
 Rod
 bacilli
 Spiral
 Spirochete and spirilla
2B: Bacteria
 Become familiar with the scientific process by
generating hypotheses, making predictions and
designing experiments
 Determine potential sources of microbial
contamination in the laboratory
 Obtain a pure culture of bacteria by streaking for
isolated colonies on solid media
 Prokaryotes: lack a nuclear membrane
 Small, single cell organisms
 Exist in huge numbers in small amounts of material
 Found almost everywhere
 Look for contamination by testing for growth on
bacterial medium.
 A population of cells, all of which are descended from
a single cell.
 Liquid or Solid
 Agar
 Non-toxic
 Remains solid at high temperatures
 Not used as a nutrient
 Defined or Complex
 To kill all living organisms
 Autoclaving
 Baking
 Alcohol
 Flaming
 Filtration
 Inoculating Loop
 Serological Pipettes
 Microliter Pipettes
 Cluster of cells visible to the naked eye
 Facilitating:
 Isolation of pure cultures
 Enumeration of cell concentration in liquid suspensions
 ID of bacterial species based upon the appearance of the
colonies
.
 A diluted suspension is pipetted directly onto the
surface of an agar plate and spread across the surface
using a sterile glass spreader.
 The number of colonies on a plate is assumed to be
equal to the number of viable cells which were spread
on the plate
 Limiting Factors
 No more than 0.2 ml of cell suspension should be spread on
the plate
 Resulting in 30 to 300 cells spread on the plate
 The number of cells per ml is directly proportional
to the mass of cells per ml
 Using Spectrophotometers to measure Optical Density
(OD)
 Light is lost as it passes through the suspension because it is
scattered and absorbed by the cells.
 Data can be used to construct a standard curve.
A Standard Curve of Viable Cells Versus Turbidity
1.5
0.5
Cells / ml
1.0E+09
8.0E+08
6.0E+08
4.0E+08
2.0E+08
0.0
0.0E+00
OD600 nm
1.0
 Positive Stains: cells pick up color
 Negative Stains: background color, cells appear white
 Labeling plates
 Label the bottom of the agar plate
 Write on the periphery of the plate
 Write your name, section, date, and location
 Use the sterile swab to collect your contaminant and
put it on the agar plate
 Incubate plates for 48 hours at 37ºC
 Isolate Pure Cultures
 Using Aseptic Technique
 E. coli
 Use Streak Techniques
 You should come to lab next with with your lab
notebook assignment almost complete.
 There will be enough time at the beginning of week 3
to observe your plates and finish the Data
Presentation/Results and Conclusions.
 Also remember to have your Pre-Lab write-up
(purpose, procedure, data table) for Exercise 3:
Enzymes at the beginning of class.