Saint Lou's School inc. General Biology 2 Laboratory ACTIVITY No. 11 PREPARATION OF BACTERIAL SMEAR AND GRAM STAINING (Introduction) Before any staining or visualization of a bacterial sample can take place, a proper smear must be prepared. A smear that is 100 thick may give a false result due to the retention of dye that should have becn rinsed away or because the thickness ‘may prevent dye penetration, A smear that is too thin may have too few cells, imcreasing the time and energy to find the bacteria under magnification. Inconclusive results due to improperly prepared slides may have an impact on patient ‘treatment and outcomes. Good smears are those which allow newsprint to be read through the smear. “The Gram stain is a diagnostic staining procedure that can be done on body fluids, tissue biopsies, throat cultures, samples from abscesses when an infection is suspected, and more. Clinically important results are obtained much more rapidly from staining than from culturing the specimen, The results of the Gram stain will aid a clinical lab in determining which ‘additional tests may be required for the identification of the bacterial strain in question. Once the bacterial gram type, ‘shape, and orientation are determined, it expedites the appropriate choice of antibiotic needed to treat the patiemt. 1. Objectives: After completing this activity, you should be able to: 1. prepare bacterial smears for the microscopic visualization of bacteria; 2. understand the chemical and theoretical basis for differential staining procedures; 3. understand the chemical basis for the Gram stain and the procedure for differentiating between two principal ‘groups of bacteria: gram-positive and gram-negative. TL Materials Cultured microorganisms from the previous activity Microscope Glass slides and coverslip Inoculating loop and needle Bunsen burner UL. Procedure A. Smears from a Broth Medium 1. Label three clean slides and number them 1, 2, and 3. Resuspend the sedimented cells in the broth culture by tapping, the culture tube with your finger. 2 With a sterile loop, place one loopful of culture on Slide 1, two loopfuls on Slide 2, and three loopfuls on Slide 3, respectively. 3. With a circular movement of the loop, spread the cell suspension into an area approximately the size of a dime. 4. Allow the slide to air-dry completely. 5. Heat fix the preparation. Note: Pass the air-dried slide through the outer portion of the Bunsen flame to prevent overheating, which can distort the morphology through plasmolysis of the cell wall. B. Smears, Solid Medium 1. Label four clean slides and label Slides 1 and 2 with an L for loop, and Slides 3 and 4 with an N for needle. 2 Using a loop, place one to two loops of water on each slide 3. With a sterile loop, touch the entire loop to the culture and emulsify the cells in water on Slide 1. Then, with a sterile ‘oop, just touch the tip of the loop to the culture and emulsify tin the water on Slide 4. Repeat Steps 1 and 2 using a sterile inoculating needle on Slides 3 and 4. 5. Allow all slides to air-dry completely 66. Heat fox the preparation Grade 11 Science. Technology. Enaineerina. and Mathematics (STEM! 20Saint Louis School Inc. ‘Bacterial Smear Preparation General Biology 2 Laboratory = / —< _ © xe one to neo loop of the cell saponin (the dean se (2) From solid medium Pace one to wo loops of water cn the contr of these ~/ @ Waar a cecuta movement ofthe oop, spre the Snpersion snto a Wan aca appronamatty the Sze ota dere lanstee 3 una amount ofthe bactnal nocubien ‘nam thesia catare eo te drop of water Spread both into a then aoa agpronmnaty te se of rack {0 Fexation for solid and broth media © Aloe ne mc 0 a dy ; < oo. gq "i O ie etn We de at reek. gy ps Sines over the fame of the Buren basta wo to ‘tece bes Part I: Gram Staining |. Gently flood smears with crystal violet and let stand for | minute. 2. Gently wash with tap water. 3. Gently flood smears with the Gram’s iodine mordant and let stand for 1 minute. 4. Gently wash with tap water. 5. Decolorize with 95% ethyl alcohol. Note: Do not over-decolorize. Add reagent drop by drop until the alcobol runs almost clear, showing only a blue tinge. Grade 17 Science, Technology, Engineering. and Mathematics STEM)_27Saint Louis School inc. General Biology 2 Laboratory 6. Gently wash with tap water 7. Counterstain with safranin for 45 seconds. 8. Gently wash with tap water. 9. Blot dry with bibulous paper and examine under oil immersion, 10. As you observe each slide under oil immersion, complete the Lab Report 6 i fh | © e105 wes ott he sta wth e's et ap wees e -_ i - - | | © cerry wasn ott he Gean'sioinn | GAH 95% secret co by de — |G Gente wash ot me HA sone 0 ap water | terete aeonot ane sont cose ah ap wate = ‘Grade 11 Science, Technology. Enaineerina, ond Mathematics (STEMI 22General Biology 2 Laboratory ‘As you observe each slide under oil immersion, complete the chart provided below. (14 pts) ‘a Draw a representative microscopic field b. Describe the cells according to their morphology and arrangement. . Describe the color ofthe stained cells. Classify the organism as to the Gram reaction: gram-positive or gram-negative. Nutrient Agar Plates Sample 1 f | 7 | | [Draw the bacterial cells viewed under / the microscope. Call color ‘Gram Reaction IV. Questions for Research ‘What are the advantages of differential staining procedures over the simple staining technique? (4 pts) 2. Cite the purpose of each of the following reagents in a differential staining procedure. (12 pts) a Primary stain . Mordant 3. Why is it essential that the primary stain and the counterstain be of contrasting colors? (5 pts) 4. Which is the most crucial step in the performance of the Gram staining procedures? Explain. (5 pts) ‘V. Documentation and Generalization (Documentation- 15 pts; Generalization- 5 pts) Grade 11 Science, Technology. Enaineerina, and Mathematics (STEMI 73