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 29-30.

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Bacterial Observation

Spherecocci

Rodbacilli

SpiralSpirochete 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 membraneSmall, single cell organismsExist in huge numbers in small amounts of

materialFound 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 SolidAgar

Non-toxicRemains solid at high temperaturesNot used as a nutrient

Defined or Complex

To kill all living organismsAutoclavingBakingAlcoholFlamingFiltration

Inoculating LoopSerological PipettesMicroliter Pipettes

Cluster of cells visible to the naked eyeFacilitating:

Isolation of pure culturesEnumeration of cell concentration in liquid

suspensionsID of bacterial species based upon the

appearance of the colonies

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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 plateLimiting 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

Positive Stains: cells pick up colorNegative Stains: background color, cells

appear white

Labeling platesLabel the bottom of the agar plate Write on the periphery of the plateWrite 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 TechniqueE. coliUse Streak Techniques

You should come to lab next with with your lab notebook assignment.

There will be enough time at the beginning of week 3 to observe your plates and finish the 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.