I.

Introduction: Microorganisms, such as bacteria and yeast, are present within all human environments without being

conspicuously recognizable to the naked human eye. Environmental swabs were taken of 11 variable locations, were isolated, and were then grown over a 48 hour incubation period in agar media for optimum colony presence. Colonies were gram stained in order to morphologically categorize bacteria by shape and gram negative or positive status.

II.

Purpose: The purpose of the laboratory was to characterize and identify bacteria on a macroscopic and

microscopic level based on the specimen of origin. Macroscopic identification was through the characterization of colonies. Microscopic identification occurred by characterizing bacteria as gram positive or negative. In addition, bacteria were characterized by form as being yeast, streptobacilli, bacilli, streptococci, staphylococci, or cocci tetrads.

III. *agar plate

Materials *crystal violet *methylene blue *safranin

*cotton swab

*distilled water *Bunsen burner *microscope slides *microscope

(counterstain) *gram iodine

*decolorizer (alcohol) *blotting pads

*inoculation loop *gloves *beaker *lighter

*dropper *oil *refrigerator *biohazard bin *air *hair *bathroom door hinge * ring *hands

*incubator * kim wipes *optical lens cleaner * paper towels *soap *lab bench

*disinfectant *doorknob *tongs

IV.

Procedures Agar Plate Inoculation and Colony Characteristics

1. Laboratory bench was disinfected and wiped down with paper towels. 2. Hands were washed and dried with paper towels. 3. 11 agar plates were selected and labeled with specimen type, date, and time during swab collection. 4. Specimens were swabbed using a cotton swab dipped with distilled water, except for tap water and mouth swab. 5. The negative control specimen was collected by turning a sealed agar plate upside down. 6. The air specimen was collected by leaving an open agar plate open exposed to the air outside of the laboratory for 30 minutes. At 30 minutes, the agar plate is sealed and turned upside down. 7. The disinfected lab bench swab is collected by wetting a cotton swab with distilled water and then dragging it across a small area of the disinfected lab bench. The cotton swab is then dragged and rotated across the agar surface lightly. The cotton swab is then disposed in the trash.

8. The hair specimen is gathered by dragging a cotton swab through a students hair from the roots to the ends. The swab is dragged and rotated lightly across the agar surface. The lid is then replaced on the agar plate. The sealed agar plate is then turned upside down. The cotton swab was wet with distilled water prior to gathering the hair specimen. 9. The unwashed hand specimen is gathered by dragging a wet cotton swab, from distilled water, between fingers and on the palm of the unwashed hands. The lid of the agar plate is removed. The specimen swab is then dragged and rotated across the topical surface of the agar. The lid is then placed back onto the agar plate, and the sealed agar plate is turned upside down. 10. A student washed their hands with antibacterial soap and then allowed them to air dry. A cotton swab, dipped into distilled water, is swabbed between the fingers and the palm. The specimen swab is then dragged and rotated across the topical surface of the agar. The lid is placed back onto the agar plate, which is then turned upside down. 11. A bathroom swab was gathered by swabbing a bathroom surface with a wet cotton swab that was dipped in distilled water. The cotton swab was then dragged and rotated across the topical surface of an agar plate. The lid of the agar plate was then replaced, and the agar plate is placed upside down. 12. A doorknob specimen was gathered by swabbing a hallway doorknob with a cotton swab that was dipped in distilled water. The specimen swab is then dragged and rotated across the topical surface of the agar and the lid is placed back onto the agar plate. The agar plate is placed upside down. 13. A mouth swab specimen was gathered by swabbing a dry cotton swab on the tongue, buccal area, and teeth of a student. The specimen swab is then dragged and rotated on a topical agar surface and the agar plate is then sealed with a lid. The agar plate is placed upside down. 14. A tap water specimen was gathered by dipping a dry cotton swab into a beaker of tap water from the sink of laboratory. The specimen cotton swab is dragged and rotated across the topical surface of an agar plate and then sealed with a lid. The agar plate is placed upside down.

15. A ring swab specimen was gathered by dragging a cotton swab wet with distilled water on the interior of a ring worn by a student. The specimen swab is then dragged and rotated across the topical surface of the agar plate and then covered with a lid. The agar plate is placed upside down. 16. All agar plate specimen smears were incubated for 48 hours at 37 Celsius to yield colony growth. 17. Agar plate specimens were then refrigerated after 48 hours to limit rate of colony growth. 18. Colonies by specimen were assessed visually through colony characteristics, such as odor, abundance of growth, color of colonies, optical traits of colonies, size, form, and elevation. Gram Staining and Microscope Identification Procedure 1. A Bunsen burner was lit and a inoculation loop was held within the flame until turning red. 2. The inoculation loop gathered a small amount of a colony within the loop and then smeared the loop onto a slide wet with a drop of distilled water. 3. The slide with bacteria was heat fixed by being quickly pulled through the Bunsen burner flame until all fluid from the slide became dry. 4. The slide was then covered by violet blue for one minute and then rinsed with distilled water. 5. The slide was then covered with iodine for 1 minute and then rinsed with distilled water. 6. The slide was then decolorized with alcohol for 15 seconds and then the alcohol was shaken off. The slide was then covered again with alcohol for another 15 seconds and then rinsed with distilled water. 7. The slide was then covered with safranin for 30 seconds and then rinsed off with distilled water. 8. The slide was blotted with the blotting pad to minimize residual water residue prior to examination under the microscope. 9. The area used for gram staining was then disinfected to wipe away residual fluids and possible bacteria. 10. The gram staining procedure was repeated for each specimen until gram stains were ready for representative colonies.

11. A microscope was cleaned with optical wipes prior to use. The microscope was plugged in. 12. A microscope was brought over and turned on at 4x magnification levels. A gram stained slide was placed on the stage and then focused via coarse and fine focuses. 13. The microscope moved to 10x magnification from 4x magnification and then fine focused. 14. The microscope moved to 40x magnification from 10x magnification and fine focused. 15. The gram stain was placed under oil immersion after 40x by placing a drop of oil onto the slide. The microscope was then moved into 100x magnification and fine focused. 16. Bacteria were noted as being gram positive (purple blue violet) or negative (red safranin) characteristics and by shape, such as coccus or bacillus. 17. The microscope identification process was repeated for all specimen types and noted under results. 18. The microscope was cleaned with optical wipes and then put away. The residual gram stains were disposed of in a biohazard bin. The general area was then disinfected and then wiped down with paper towels. 19. Participants disposed of gloves into the trash and then washed their hands. V. Results Please refer to table 1: Bacteria Specimen Assessment VI. Discussion:

All agar plates smeared with specimens resulted in colony growth after a 48 incubation period leading to a supporting conclusion that bacteria is present within a wide spectrum of environmental samples. The negative control yielded a slight abundance of growth with small yellow and white colonies. The air sample specimen grew large yellow and moderate white sized colonies with slight abundance of growth. The air sample and the negative control had similar representative colonies based on color and abundance of growth. This may represent a possibility that the agar plate may have been contaminated within the lab, during manufacture, during handling, or by simple airflow. In addition, the negative control and air sample were both similar in terms of metabolic byproducts evaluated on the basis of colony pigmentation. Thus, the negative control may

have had air filtration into the agar plate leading to an introduction of bacteria onto the agar as a localized explanation of cross contamination. The disinfected lab bench grew green, yellow, white, and orange colonies with a slight abundance of growth. Gram-positive staphylococci bacteria were detected in yellow, white, and orange colonies that were all small in size. The large green colony was characterized as having the presence of yeast. The bathroom specimen had an increasingly large abundance of growth amongst small yellow, small beige with red center, and large beige colonies. All colonies had detectable gram-positive staphylococcus bacteria within the bathroom specimen. The characteristic of a large abundance of growth was seen amongst the bathroom, ring, and washed hands. This may be related to the high amount of contact or an alternative close proximity to human surfaces that a bathroom, a ring, and hand may have resulting in a higher presence of bacteria. The unwashed hand specimen displayed larger colony abundance of growth in comparison to the washed hands specimen, although colony pigmentation and bacterial presence remained the same. This may have been affected by a number of variables affecting efficacy of hand washing, such as water flow, water temperature, bacterial resistance to soap, and introduction of additional bacteria from tap water. Water temperature during hand washing is close to body temperature, or 37 C, which is also the optimum incubation temperature for bacterial colonies. Insufficient hand scrubbing or water flow may inadequately remove debris such as keratinized epithelial cells of the epidermal apical surface of the stratum corneum, fats, dirt, and bacteria. The use of air drying over paper towel hand drying may have also inadequately removed bacteria from the hands. Overall, the majority of bacteria present amongst specimens were gram positive cocci subtypes. Specimens with moderate abundance of growth were characteristically associated with human contact or proximity, such as the doorknob, unwashed hands, and hair. In this regard, abundance of growth was correlated to proportional exposure and proximity to contact, which is highlighted by the principle of fomite aided transmission.