LAB PRACTICAL 1

Lab safety: 

• Know where the fire extinguisher, fire blanket, and first aid kit are kept. Under the professor’s desk. 

• What is the solution used to clean your lab bench at the beginning and end of each lab class? 70% ethanol. 

• Where do you dispose of the following items: wet mounts go in the bleach bath, test tubes go in the kill rack, slides that have been heat fixed or any other glass items goes in the sharp's container, used pipettes, cotton swabs, or Petri dishes goes in the kill bucket or biohazard can. 

Lab 1 Environmental Sampling: 

• What is the mound of cells found on a petri dish called? Colony 

• How many bacterial cells does it take to start this mound? One bacterial cell. 

• How many bacterial cells are found in this mound? One trillion. 

• Why are Petri dishes incubated upside down? To avoid condensation on growth. 

• Why are Petri dishes incubated at 37^oC? Body temperature is the favorable growth conditions for microorganisms. 

Lab 2 Microscope and Morphology: 

• Be able to label and state the function of these microscope parts: eyepiece houses ocular lens, condenser collects and directs light from light source to slide, stage supports the slide, diaphragm regulates the amount of light that gets to the slide, revolving nosepiece changes magnifications, stage control knobs positions slide to change field of view, slide/stage clips holds slides in place. 

• Where are your two hands placed when carrying the microscope? Arm and base. 

• Be able to label the coarse and fine adjustment knobs. Know which knob is used when focusing with each of these objective lenses: scanning lens is red, low magnification lens is yellow, high dry lens is blue, and oil immersion lens. 

• Which lens is first used when focusing on a specimen? Which lens is clicked in place before storing? Scanning lens. 

• Determine the total magnification depending upon which objective lens is facing down. Scanning power lens (4x): 4 x 10= 40x and low magnification lens (10x): 10 x 10 = 100x High dry lens (40x): 40 x 10= 400x  Oil immersion lens (100x): 100 x 10= 1000x 

• When storing the scope, is the stage in the up or down position? The stage is down. 

• Be able to state the bacterial shape when viewing a specimen under a microscope: coccus, bacillus, or spirillum. Coccus-circle, bacillus-rods, spirillum-rigid spiral. 

Lab 3 Aseptic Technique, Smear Prep, and Simple Stain: 

• What dye was used in the simple stain? Methylene blue 

• What is the charge on bacterial cells? -negative. On the dyes we use in lab? -positive. 

• What is the purpose of aseptic technique? To avoid contamination. 

• Where is the target circle drawn on the slide when making a smear prep? On the back of the slide. 

• Which tool is used to transfer the bacteria when making a smear prep? Inoculating loop. 

• Be able to name one of the two items flamed when performing aseptic technique. Open test tube, inoculating loop. 

• What is the important step done after air drying the specimen? What are the two purposes of this step? Heat fix the slide to kill the microbes and make them stick to the slide. 

Lab 4 Gram Stain and Cell Model: 

• Which molecule in the bacterial cell is targeted in the Gram stain? Peptidoglycan. Where is it found in the bacterial cell? On the cell wall. Know how the molecule varies if the bacterial cell is Gram positive or Gram negative. Gram positive; thick peptidoglycan; stains purple. Gram negative peptidoglycan; thin peptidoglycan; stains pink/red. 

• Be able to identify a Gram positive and Gram-negative specimen under the microscope.  

• Know the primary dye, decolorizer, and counterstain. Primary dye; crystal violet, Decolorizer; 95% ethanol, counterstain; safranin. 

• Be able to correctly write one bacterial species that is Gram positive and one that is Gram negative. Gram positive; staphylococcus aureus, streptococcus agalactiae, mycobacterium smegmatis, bacillus cereus. Gram negative; Escherichia coli, klebsiella pneumoniae. 

• Be able to give an error for a Gram-positive cell turning out Gram negative (pink). Over decolorizing. 

• Be able to give an error for a Gram-negative cell turning out Gram positive (purple). Under decolorizing. 

• Be able to state the bacterial arrangement when viewing a specimen under a microscope: staphylococcus-staphylococcus, streptococcus-streptococcus, or streptobacillus-streptobacillus. Be able to name one species for each arrangement. 

• For the bacterial cell model, be able to label the flagellum, fimbriae, ribosome, plasmid, and DNA or chromosome 

• Know the region of the bacterial cell where the chromosome is found. Nucleoid region 

• Be able to recognize the endospore on the model. 

• For the two cell wall models, be able to identify which is the Gram positive and which is Gram negative. For both be able to label the layers: cell membrane, peptidoglycan, and outer membrane (only on Gram negative) 

Lab 5 Acid Fast Stain: 

• Which molecule in the bacterial cell is targeted in the acid-fast stain? Mycolic acid. 

• Does this technique use steam made through the boiling water bath? Yes. 

• Be able to identify an acid fast positive and acid-fast negative specimen under the microscope. Acid fast positive-pink/red, acid fast negative-blue. 

• Know the primary dye, decolorizer, and counterstain. Carbolfuchsin, acid acholol, methylene blue. 

• Be able to correctly write the bacterial species that is acid fast positive and one that is acid fast negative. Mycobacterium smegmatis. 

• Be able to give an error for an acid-fast positive cell turning out acid fast negative. - over washed with acid alcohol or did not use enough steam or heat. 

Lab 6 Endospore/Spore Stain: 

• Does this technique use steam made through the boiling water bath? Yes. 

• Be able to identify an endospore positive and endospore negative specimen under the microscope. Endospore positive-pink rods and green ovals and endospore negative-see all pink. 

• Know the primary dye and counterstain. Is there a decolorizer? Primary dye: malachite green and counterstain: safranin. NO DECOLORIZER. 

• Be able to correctly write one bacterial species that is endospore/spore positive and one that is endospore/spore negative. Bacillus cereus is endospore positive and endospore negative is mycobacterium smegmatis. 

• Be able to label the pink and green structures found in the spore stain. Pink=vegetative cells and green=endospores. 

• Be able to give an error for an endospore positive cell turning out endospore negative (spores did not stain). Not enough steam or heat. 

Lab 7 Motility Testing: 

• Know the structure the bacterial cell has if it is motile. flagella 

• Know the movement associated with a motile and nonmotile species when viewed under a wet mount. Motile: straight run and tumble, and nonmotile; Brownian motion. 

• Be able to name one bacterial species used in lab that is motile and nonmotile. Escherichia coli and bacillus cereus.  

• What tool is used for the stab tube motility test? Inoculating needle. 

• What is the physical property of the media used for the motility test? Semi solid. 

• If given a stab tube motility test, be able to state if it is a motile or nonmotile result. 

• Be able to state the error if a non-motile species appeared motile on the stab tube. Not going straight in and out or wiggled needle.