Acne Medication Testing

Introduction to Acne

Acne is a common chronic skin condition characterized by the inflammation of hair follicles and sebaceous (oil) glands. This condition arises when hair follicles become clogged with oil (sebum) and dead skin cells, and it is most prevalent during adolescence but can occur at any age. Acne manifests in various forms, including pimples, blackheads, whiteheads, cysts, and nodules. Pimples, which can be either papules or pustules, appear as small, raised, red, and tender bumps on the skin. Papules lack pus, while pustules have a white or yellow pus-filled center. Blackheads, or open comedones, are small, dark-colored spots on the skin surface resulting from the oxidation of melanin when the clogged pore is exposed to air. Whiteheads, or closed comedones, are small, flesh-colored or white bumps that form when hair follicles are clogged with sebum and dead skin cells but remain closed at the skin surface. Cysts and nodules are severe forms of acne; cysts are large, painful, pus-filled lesions deep within the skin, while nodules are solid, painful lumps beneath the skin surface. Both can cause scarring.

The primary causes of acne include excess sebum production, follicular hyperkeratinization, bacterial involvement, hormonal changes, genetic factors, and environmental factors. Overactive sebaceous glands produce too much oil, which mixes with dead skin cells to clog pores. Hormonal changes, particularly during puberty, menstruation, and pregnancy, can increase sebum production. Follicular hyperkeratinization, an increase in the shedding of skin cells around hair follicles, can lead to clogged pores, influenced by genetic predisposition and hormonal fluctuations. Cutibacterium acnes (C. acnes) bacteria, which naturally live on the skin, multiply in clogged pores, causing inflammation and pus formation. Conditions that promote bacterial growth, such as excessive oil production and lack of oxygen in clogged pores, exacerbate this issue. Hormonal fluctuations, particularly androgens, can increase sebum production and lead to acne, triggered by puberty, menstrual cycles, pregnancy, and conditions like polycystic ovary syndrome (PCOS). Genetic factors play a role, as a family history of acne increases the likelihood of developing the condition. Environmental factors such as diet, stress, and exposure to certain chemicals or cosmetics can exacerbate acne, with high-glycemic diets, dairy consumption, stress, and use of oily or comedogenic products being common triggers.

The concerns associated with acne extend beyond its immediate symptoms. Severe acne, particularly cystic and nodular acne, can lead to permanent scarring, including icepick, boxcar, and rolling scars. Early and effective treatment is crucial to prevent long-term skin damage. Post-inflammatory hyperpigmentation (PIH) occurs when healed acne leaves dark spots on the skin, and managing this requires sun protection and topical treatments like retinoids and vitamin C. Acne can significantly affect self-esteem, leading to emotional distress, anxiety, and depression. Psychological support and counseling, alongside medical treatment, can help individuals cope with the emotional impact of acne. Additionally, acne is often a chronic condition that can persist for years, requiring ongoing management. Regular skincare routines, appropriate use of medications, and lifestyle modifications are essential for long-term control. Understanding acne’s definitions, appearances, causes, and concerns is crucial for effective management and treatment. By addressing the underlying causes and taking preventive measures, individuals can achieve better skin health and reduce the impact of acne on their lives.

Causes of Acne

Excess Sebum Production

Sebaceous glands are responsible for producing sebum, an oily substance that helps protect and moisturize the skin. This sebum is essential for maintaining the skin’s barrier function and preventing it from becoming dry and brittle. However, overproduction of sebum can lead to clogged pores, which is a primary factor in the development of acne. Excess sebum can mix with dead skin cells and other debris, creating an environment conducive to the formation of comedones (blackheads and whiteheads) and inflammatory acne lesions.

Follicular Hyperkeratinization

Follicular hyperkeratinization refers to an abnormal increase in the shedding of skin cells around hair follicles. Normally, skin cells are shed and replaced at a steady rate, but in individuals with acne, this process can become accelerated and disordered. The excess shedding of skin cells can combine with sebum to block hair follicles, creating a plug that traps oil and bacteria inside. This blockage is a critical step in the formation of acne, as it provides a breeding ground for bacteria and triggers an inflammatory response.

Bacterial Involvement: Acne Vulgaris

The bacterium C. acnes naturally resides on the skin and plays a significant role in the development of acne vulgaris, the most common form of acne. Under normal circumstances, C. acnes is part of the skin’s microbiome and does not cause harm. However, when pores become clogged with sebum and dead skin cells, the anaerobic conditions inside the clogged follicle allow C. acnes to proliferate. This overgrowth of bacteria leads to inflammation and the formation of acne lesions, including papules, pustules, nodules, and cysts.

Hormonal Changes

Hormonal fluctuations are a well-known trigger for acne, particularly during life stages such as puberty, menstruation, pregnancy, and periods of high stress. Androgens, a group of hormones that includes testosterone, can increase the size and activity of sebaceous glands, leading to increased sebum production. During puberty, both males and females experience a surge in androgen levels, which explains the high prevalence of acne during this period. Similarly, hormonal changes associated with the menstrual cycle, pregnancy, and conditions like polycystic ovary syndrome (PCOS) can also trigger or worsen acne.

Genetic Factors

Genetics play a significant role in determining an individual’s susceptibility to acne. A family history of acne can increase the likelihood of developing the condition, suggesting a genetic predisposition. Genetic factors can influence various aspects of acne development, including sebum production, skin cell turnover, inflammatory response, and the skin’s microbiome. Understanding these genetic influences can help in identifying individuals at higher risk and tailoring more effective treatment strategies.

Environmental Factors

Several environmental factors can exacerbate acne, including diet, stress, and exposure to certain chemicals or cosmetics. A diet high in refined sugars and dairy products has been linked to increased acne severity, although the exact mechanisms are still being studied. Stress can exacerbate acne by triggering hormonal changes and increasing inflammation. Additionally, the use of certain cosmetics, skincare products, or chemicals that are comedogenic (pore-clogging) can worsen acne. Environmental pollutants and humidity can also contribute to the condition by increasing sebum production and promoting bacterial growth.

Effects of Acne

Non-inflammatory Lesions: Comedones

·         Blackheads (Open Comedones): Blackheads are small, dark-colored spots on the skin surface. They occur when pores become clogged with sebum and dead skin cells, which are then exposed to air. The dark appearance is due to the oxidation of melanin in the clogged material, not dirt as commonly believed. Blackheads are typically found on the nose, forehead, and chin.

·         Whiteheads (Closed Comedones): Whiteheads are small, flesh-colored or white bumps that form when hair follicles are clogged with sebum and dead skin cells but remain closed at the skin surface. Unlike blackheads, whiteheads are not exposed to air and therefore do not oxidize and turn dark. They commonly appear on the face, shoulders, chest, and back.

inflammatory Lesions: Papules, Pustules, Nodules, Cysts

Papules are small, raised, red, and tender bumps on the skin. They do not contain pus. Papules form when hair follicles become inflamed due to a combination of sebum, dead skin cells, and bacteria. The inflammation causes the follicle walls to break down, leading to the formation of these red lesions.

Pustules are similar to papules but have a white or yellow pus-filled center, making them appear like typical pimples. Pustules form when the inflammation from a papule leads to a build-up of pus, which consists of white blood cells, bacteria, and dead cells. This type of acne is often more painful and prominent than papules.

Nodules are large, solid, and painful lumps located deep beneath the surface of the skin. Nodules develop when the clogged and inflamed hair follicles undergo further irritation and infection. Unlike pustules, nodules are deep within the skin and are not filled with pus. They can be very painful and are prone to causing scarring.

Cysts are deep, painful, pus-filled lesions that are larger than pustules and nodules. Cysts form when a severe inflammatory response occurs deep within the skin. The infection and inflammation can lead to significant swelling and pain. Cystic acne is considered the most severe form of acne and is most likely to cause scarring.

Lasting Effects of Acne

Scarring occurs when the skin’s collagen structure is damaged during the healing process of severe acne lesions, particularly nodules and cysts. The body attempts to repair the damage by producing collagen, but this process can be imperfect, leading to scar formation.

·         Icepick scars are deep, narrow, and pitted scars that resemble puncture marks. They penetrate deeply into the skin, giving the appearance of being "ice-picked."

·         Boxcar scars are broad, rectangular depressions with sharply defined edges. They are often wider than icepick scars and give the skin an uneven appearance.

·         Rolling scars create a wave-like, undulating appearance on the skin due to bands of scar tissue that form under the surface. They are typically broader and shallower than boxcar and icepick scars.

Post-Inflammatory Hyperpigmentation (PIH) is a condition where healed acne lesions leave dark spots or patches on the skin. These dark spots are not scars but are caused by an excess production of melanin (the pigment that gives skin its color) in response to inflammation. PIH can vary in color from pink to red, purple, brown, or black, depending on skin tone and the severity of the inflammation. PIH occurs as the skin’s natural response to inflammation. When an acne lesion heals, the inflammation can stimulate melanocytes (pigment-producing cells) to produce more melanin, resulting in dark spots that can persist for months or even years.

Acne can significantly affect an individual's self-esteem and body image. The visibility of acne on the face and other prominent areas can lead to feelings of self-consciousness and embarrassment. The persistent and chronic nature of acne can lead to emotional distress, including frustration, anxiety, and feelings of hopelessness. In severe cases, acne can contribute to mental health issues such as anxiety disorders and depression. The social stigma and perceived judgment from others can exacerbate these feelings. Acne can affect social interactions and relationships, with individuals sometimes avoiding social situations due to their appearance. This can lead to social isolation and a diminished quality of life.

Prevention of Acne

It is crucial to have a proper skincare routine and choose skincare products labeled as "non-comedogenic," which means they are formulated to not clog pores. Regular cleansing helps remove excess oil, dirt, and sweat, which can contribute to clogged pores. Washing the face twice a day—once in the morning and once in the evening—is typically sufficient. Additionally, cleansing the skin after activities that cause sweating, such as exercise, can help prevent acne. Gentle cleansers help remove excess oil, dirt, and makeup without stripping the skin of its natural moisture. Non-comedogenic moisturizers help keep the skin hydrated without contributing to pore blockages. Scrubbing the skin harshly or washing it too frequently can irritate and damage the skin, leading to increased inflammation and oil production. Gentle cleansing, typically twice a day, is sufficient to keep the skin clean without exacerbating acne. Makeup and skincare products that are oil-free and labeled as non-comedogenic are less likely to clog pores and contribute to acne. It's essential to read product labels carefully and select formulations that suit acne-prone skin. Avoiding heavy, greasy products can help prevent the formation of comedones.

Maintain a balanced diet, manage stress, and avoid excessive touching of the face. A balanced diet rich in fruits, vegetables, whole grains, and lean proteins can support overall skin health. Some studies suggest that high-glycemic foods and dairy products may exacerbate acne, so it may be beneficial to monitor and adjust dietary habits accordingly. Stress can trigger hormonal changes that increase sebum production and inflammation, leading to acne flare-ups. Stress management techniques such as exercise, meditation, and adequate sleep can help reduce the impact of stress on the skin. Frequent touching of the face can transfer oils, dirt, and bacteria from the hands to the skin, potentially clogging pores and causing acne. It's important to minimize touching the face and to always wash hands before applying skincare products or makeup.

Protecting the skin from UV damage is essential, as sun exposure can worsen acne and cause post-inflammatory hyperpigmentation. Using a broad-spectrum sunscreen that is non-comedogenic and suitable for acne-prone skin can help prevent further skin damage without clogging pores. Look for sunscreens that are oil-free and specifically formulated for sensitive or acne-prone skin.

Acne Treatments

Topical Treatments

Benzoyl peroxide works by killing bacteria, which are a major contributor to acne. It also helps to remove excess oil and dead skin cells from the skin's surface, preventing the formation of clogged pores. Benzoyl peroxide is available over-the-counter in various strengths and formulations, including gels, creams, and cleansers. It is typically applied once or twice daily to the affected areas. It's important to start with a lower concentration to minimize skin irritation and gradually increase as tolerated.

Topical retinoids (Tretinoin, Adapalene) promote cell turnover and prevent the formation of clogged pores. They work by increasing the rate at which skin cells are shed and replaced, which helps to clear existing acne and prevent new lesions from forming. Retinoids also reduce inflammation associated with acne. Topical retinoids are usually applied once daily, typically at night, to the affected areas. They can cause initial irritation and increased sensitivity to sunlight, so it's important to use sunscreen during the day and start with a lower concentration.

Topical antibiotics (Clindamycin, Erythromycin) reduce the populations of bacteria on the skin and decrease inflammation. By targeting the bacterial component of acne, they help to reduce the severity and number of acne lesions. Topical antibiotics are often combined with other treatments, such as benzoyl peroxide or topical retinoids, to prevent antibiotic resistance and enhance efficacy. They are typically applied once or twice daily to the affected areas.

Salicylic acid is a beta-hydroxy acid (BHA) that helps exfoliate the skin and clear pores. It penetrates the pores and helps dissolve the debris and dead skin cells that can cause blockages, thereby preventing and treating acne. Salicylic acid is available over-the-counter in various forms, including cleansers, toners, and spot treatments. It is typically used once or twice daily, depending on the product and individual skin tolerance.

Oral Medications

Oral antibiotics (Doxycycline, Minocycline) work by reducing the bacterial load of P. acnes and decreasing inflammation from within the body. They target the bacteria that contribute to acne and help to calm the inflammatory response associated with acne lesions. Oral antibiotics are typically prescribed for moderate to severe acne and are used for a limited duration to minimize the risk of antibiotic resistance. They are often combined with topical treatments for optimal results.

Oral contraceptives regulate hormones that contribute to acne formation, particularly androgens that increase sebum production. By balancing hormone levels, they can help reduce acne in females. Oral contraceptives are prescribed for females with hormonal acne. They are taken daily, following the prescribed regimen, and can also provide other benefits, such as regulating menstrual cycles. Spironolactone is an anti-androgen medication that reduces androgen levels, thereby decreasing sebum production. It helps to balance hormonal fluctuations that can trigger acne. Spironolactone is often prescribed to females with hormonal acne. It is taken orally, and the dosage is adjusted based on the individual's response and any side effects. It is particularly useful for treating acne associated with hormonal conditions like polycystic ovary syndrome (PCOS).

Isotretinoin (Accutane) is a potent retinoid that significantly reduces sebum production, unclogs pores, and decreases inflammation. It works by shrinking the sebaceous glands and altering the skin's oil production. Isotretinoin is reserved for severe, cystic acne that is unresponsive to other treatments. It requires close monitoring by a healthcare professional due to potential side effects, including dryness, liver function changes, and teratogenicity (risk of birth defects). It is typically taken for a course of 15-20 weeks.

Lab Exercise – Course-Based Ungergraduate Research Experience (CURE)

Objective

The objective of this lab is to investigate the impact of various acne medications on facial bacteria. The lab aims to assess the growth characteristics and responses of bacteria collected from the skin to different acne medications. The experiment involves the inoculation of sterile broth tubes and agar plates with facial bacteria, followed by incubation and observation of bacterial growth.

The first step involves collecting facial bacteria using sterile swabs and inoculating them into broth tubes. These broth tubes provide a nutrient-rich environment for bacterial growth. After inoculation, the tubes are securely closed and incubated at the appropriate temperature for bacterial growth. The incubation period allows the bacteria to multiply and adapt to the conditions. Simultaneously, agar plates are prepared and labeled. The agar serves as a solid medium for bacterial growth and allows for the visualization of distinct colonies. The plates are inoculated using aseptic technique, ensuring that a small sample of bacteria from the broth tube is streaked evenly across the agar surface. The control plate serves as a baseline for comparison, while the other plates are inoculated with the bacteria and treated with specific acne medications.

Different identification tests, such as Gram staining, Mannitol Fermentation, Bile Tolerance and Esculin Hydrolysis, Hemolysis on Blood Agar, Triple Sugar Iron (TSI) Slants, Methyl Red-Voges-Proskauer (MRVP) Broth, Nitrate Reduction Broth, Sulfide Indole Motility (SIM) Deeps, Citrate Plates, Oxidase Test, and Catalase Test, are performed to characterize and classify the bacterial species present. After incubation, the plates are observed for bacterial growth and any visible differences in colony morphology and appearance. The growth characteristics, such as colony size, color, texture, transparency, and form, are recorded. Additionally, a representative colony from each plate is subjected to a Gram stain, allowing for the examination of cell shape, arrangement, and staining reaction.

Identification tests will be conducted to determine the species of the bacteria. The lab also focuses on analyzing the effects of acne medications on bacterial growth by comparing the growth characteristics and test outcomes between the plates with different medications and the control plate. Through data analysis, the lab aims to draw conclusions about the efficacy of acne medications in inhibiting bacterial growth and inform potential treatment options.

The data collected is analyzed to evaluate the effects of acne medications on bacterial growth. By comparing the growth characteristics and test outcomes between the plates with different medications and the control plate, conclusions can be drawn regarding the efficacy of the acne medications in inhibiting bacterial growth and potentially informing treatment options. Throughout the lab, it is important to maintain accurate records, adhere to proper aseptic techniques, and consider potential limitations or sources of error that may affect the interpretation of the results.

Materials

·         Clean and sanitized workspace

·         Personal protective equipment (PPE) such as gloves and lab coat

·         Sterile broth tubes

·         Sterile swabs

·         Labeling materials (e.g., marker)

·         Incubator set to the appropriate temperature for bacterial growth

·         TSA plates (appropriate number)

·         Acne medications (e.g., Medication A, Medication B, Medication C, Medication D)

·         Additional materials or reagents required for identification tests (as mentioned in the Identification Lab section)

·         Sterile inoculating loops

·         Bacterial culture media (e.g., nutrient agar, Mannitol Salt Agar, Bile Esculin Agar, Blood Agar, TSI slants, MRVP broth, nitrate reduction broth, SIM deeps, citrate plates)

·         Crystal violet

·         Iodine solution

·         Ethanol

·         Safranin

·         Filter paper or oxidase test strips

·         Oxidase reagent (e.g., N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride)

·         Hydrogen peroxide (H2O2)

·         Rabbit plasma

·         Bergey's Manual of Determinative Bacteriology (for bacterial identification)

·         Microscope with oil immersion lens

·         Zinc powder (for nitrate reduction test)

·         Distilled water

Note: It is essential to maintain aseptic technique throughout the protocol to prevent contamination. Adhere to appropriate safety guidelines and dispose of used swabs and other materials properly.

Protocol

Facial Bacteria Inoculation

1.       Preparation

a.       Ensure that the workspace is clean and sanitized.

b.       Put on appropriate personal protective equipment (e.g., gloves, lab coat).

c.        Label the sterile broth tubes with the appropriate identification, including student names and date.

2.       Skin Bacteria Collection

a.       Open a sterile swab package without touching the swab tip or inner part of the package.

b.       Gently swab the facial area of the skin using a circular motion for approximately 10 seconds, ensuring contact with the skin surface.

3.       Inoculation of Broth

a.       Holding the labeled broth tube in one hand, open the tube use aseptic technique.

b.       Insert the swab into the broth tube, ensuring that the swab tip is immersed in the broth.

c.        Rotate the swab inside the broth tube several times to dislodge and transfer the bacteria into the broth.

d.       Remove the swab from the broth tube while avoiding contact with the tube's rim or inner surface.

e.       Securely close the broth tube to prevent contamination.

f.         Gently mix the broth tube by inverting it several times to ensure even distribution of the collected bacteria.

4.       Incubation

a.       Place the inoculated broth tubes in an incubator set to the appropriate temperature for bacterial growth.

b.       Ensure that the tubes are placed upright and securely in the incubator.

c.        Incubate the broth tubes for the specified duration, typically 24 to 48 hours, depending on the bacterial species of interest.

Inoculating the Plates

1.       Preparation

a.       Ensure that the workspace is clean and sanitized.

b.       Put on appropriate personal protective equipment (e.g., gloves, lab coat).

c.        Label the TSA plates with the appropriate identification, including the plate number and medication name.

d.       Prepare any additional materials or reagents required for subsequent steps, as mentioned in the Identification Lab.

2.       Inoculation of Plates

a.       Label 5 plates with name, date, then label one control, then in sequence Medication A, Medication B, Medication C, Medication D.

b.       Hold the labeled TSA plate for the control group with one hand, keeping it close to the working area.

c.        Using a sterile inoculating loop, obtain a small sample of the bacteria from the inoculated broth tube containing the facial bacteria.

d.       Starting from one edge of the TSA plate, streak the inoculating loop across the surface of the agar using a back-and-forth motion while rotating the plate.

e.       After streaking, close the control plate promptly to minimize contamination risk.

f.         Repeat steps c-e for each medication plate, using a sterile inoculating loop and a new sample from the inoculated broth tube.

3.       Application of Acne Medications

a.       Label the medication tubes or containers with the appropriate identification.

b.       Using a sterile inoculating loop, obtain a small amount of Medication A from its container.

c.        Gently streak the Medication A in a straight line on the surface of the TSA plate inoculated with Medication A, perpendicular to the bacterial streak.

d.       Repeat steps b and c for each medication, ensuring that each medication is applied only to its corresponding plate.

e.       Close the medication containers promptly to prevent contamination.

4.       Incubation

a.       Place all the inoculated and medicated plates, including the control and acne medication plates, in an incubator set to the appropriate temperature for bacterial growth.

b.       Ensure that the plates are securely closed and properly labeled.

c.        Incubate the plates for the recommended duration, typically 24 to 48 hours, at the specified temperature.

5.       Record Keeping

a.       Maintain a detailed record of the plate numbers, corresponding medications, and any other relevant information.

b.       Note the date and time of inoculation.

Culture Analysis

1.       After incubation, observe the plates for bacterial growth and record any differences in colony morphology and appearance.

a.       Document the growth characteristics of each plate, including colony size, color, texture, transparency, and form.

2.       Perform a Gram stain on a representative colony from each plate and observe the staining reaction, cell shape, and arrangement. (procedure below)

a.       Prepare a bacterial smear from a colony on the control plate and a colony within the zone of inhibition on each medicated plate.

b.       Allow the smears to air dry completely.

c.        Apply a few drops of Crystal violet to each smear and let it sit for 1 minute.

d.       Gently rinse the smears with distilled water.

e.       Apply a few drops of Iodine solution to each smear and let it sit for 1 minute.

f.         Rinse the smears again with distilled water.

g.       Decolorize the smears by gently flooding them with ethanol until the runoff is colorless.

h.       Rinse the smears with distilled water.

i.         Counterstain the smears by applying a few drops of Safranin and letting it sit for 1 minute.

j.         Rinse the smears with distilled water and allow them to air dry.

k.        Observe the stained smears under a microscope using oil immersion.

Isolation of Bacteria from Control Plate

1.       Label a sterile nutrient agar plate as "Control Isolate”.

2.       Using a sterile inoculating loop, carefully streak the surface of the control plate, picking up a small amount of bacterial growth.

3.       Perform quadrant streaking isolation on the new plate with your sample collected.

4.       Incubate the control plate isolate plate(s) upside down in an incubator set to the appropriate temperature for the bacterial growth for 24-48 hours.

5.       Pick individual colonies after incubation and subculture them in a nutrient broth.

6.       Repeat this process for as many isolations as needed.

Isolation of Bacteria from Zone of Inhibition

1.       Label sterile nutrient agar plates as "Med A Isolate," "Med B Isolate," and so on, depending on the number of isolates to be tested.

a.       Growth from Medicine A plate goes on Med A Isolate plate, and so on.

2.       Using a sterile inoculating loop, gently scrape the bacterial growth in the zone of inhibition on the respective plate.

3.       Perform quadrant isolation on the new plate with your sample collected.

4.       Incubate the control plate isolate plate(s) upside down in an incubator set to the appropriate temperature for the bacterial growth for 24-48 hours.

5.       Pick individual colonies after incubation and subculture them in a nutrient broth.

6.       Repeat this process for as many isolations as needed.

Identification of Bacteria

1.       Based on the Gram stain results, perform follow-up tests.

2.       Refer to Bergey's Manual of Determinative Bacteriology to identify the Major Category, Group, and Genus of the bacteria based on observed characteristics and test outcomes.

3.       Perform additional tests as necessary to differentiate among species and determine the species of the bacteria.

a.       Use Bergey's Manual of Systematics of Archaea and Bacteria to confirm results and discover follow-up tests.

Mannitol Fermentation

1.       Prepare a Mannitol Salt Agar (MSA) plate.

2.       Streak the bacterial isolate onto the surface of the MSA plate using a sterile inoculating loop.

3.       Incubate the plate at the appropriate temperature for the bacterial isolate for 24-48 hours.

1.       Observe the growth of the bacteria on the MSA plate and check for a change in the color of the agar surrounding the bacterial growth.

a.       Acid production from mannitol fermentation may result in a yellow color change.

Bile Tolerance and Esculin Hydrolysis

1.       Prepare a Bile Esculin Agar (BEA) plate.

2.       Streak the bacterial isolate onto the surface of the BEA plate using a sterile inoculating loop.

3.       Incubate the plate at the appropriate temperature for the bacterial isolate for 24-48 hours.

4.       Examine the growth of the bacteria on the BEA plate.

a.       Bile tolerance is indicated by the presence of bacterial growth

b.       Esculin hydrolysis is evidenced by a blackening of the medium due to the breakdown of esculin.

Hemolysis on Blood Agar

1.       Prepare a Blood Agar plate.

2.       Streak the bacterial isolate onto the surface of the Blood Agar plate using a sterile inoculating loop.

3.       Incubate the plate at the appropriate temperature for the bacterial isolate for 24-48 hours.

4.       Examine the growth of the bacteria on the Blood Agar plate.

a.       Hemolysis may manifest as

                                                               i.      Complete clearing (beta-hemolysis)

                                                             ii.      Partial clearing (alpha-hemolysis)

                                                            iii.      No clearing (gamma-hemolysis) around the bacterial growth, also known as (non-hemolytic)

Triple Sugar Iron (TSI) Slants

1.       Inoculate a TSI slant by stabbing the bacterial isolate into the center of the slant using a sterile inoculating needle, then streaking the slant after the needle is pulled out.

2.       Incubate the TSI slant at the appropriate temperature for the bacterial isolate for 18-24 hours.

3.       Interpreting the TSI test results involves analyzing the color changes and growth patterns observed in both the slant and butt regions of the agar.

a.       Slant color:

                                                               i.      Red: If the slant remains red, it indicates that no fermentation of any of the three sugars has occurred. This result is denoted as K or A (K for alkaline, A for acid).

                                                             ii.      Yellow: A yellow slant indicates the fermentation of one or more sugars, resulting in an acidic pH. This result is denoted as A/A (A for acid).

                                                            iii.      Red at the top, yellow at the bottom: This result, known as K/A, suggests that the bacteria have fermented glucose only, producing acid in the butt region but not in the slant region.

b.       Butt color:

                                                               i.      Yellow: A yellow butt indicates fermentation of glucose, lactose, or sucrose, resulting in an acidic pH. This result is denoted as A/A (A for acid).

                                                             ii.      Red: A red butt suggests that no fermentation of any of the sugars has occurred. This result is denoted as K or A (K for alkaline, A for acid).

                                                            iii.      Black precipitate: The presence of a black precipitate in the butt indicates hydrogen sulfide (H2S) production, resulting from the breakdown of sulfur-containing amino acids. This result is denoted as (H2S+).

                                                            iv.      Cracks or lifting of the agar: Gas production by the bacteria may cause cracks or lifting of the agar. This result is denoted as gas positive (G+). Record the results and interpret the metabolic reactions based on the observed changes in the slant and butt regions.

Methyl Red-Voges-Proskauer (MRVP) Broth

1.       Inoculate a tube of MRVP broth with the bacterial isolate using a sterile inoculating loop.

2.       Incubate the MRVP broth at the appropriate temperature for the bacterial isolate for 24-48 hours.

3.       After incubation, divide the MRVP broth into two separate tubes, one labeled MR and the other VP.

4.       Perform the MR test on one tube by adding a few drops of methyl red indicator to the broth and observe for a color change.

a.       Red color, indicates a positive result for mixed acid fermentation.

5.       Perform the VP test on the other tube by adding VP reagents (alpha-naphthol and potassium hydroxide) and observe for the color development

a.       Red color, indicates a positive result for butanediol fermentation.

Nitrate Reduction Broth

1.       Inoculate a tube of nitrate reduction broth with the bacterial isolate using a sterile inoculating loop.

2.       Incubate the nitrate reduction broth at the appropriate temperature for the bacterial isolate for 24-48 hours.

3.       After incubation, add a few drops of nitrate reagents A and B to the broth.

4.       Observe for a color change in the broth.

a.       If a red color develops, it indicates the reduction of nitrate to nitrite.

b.       If there is no color change, proceed to the next step.

5.       Add a small amount of zinc powder to the broth.

6.       Observe for a color change.

a.       If a red color develops after the addition of zinc, it indicates the presence of nitrate, indicating a negative result for nitrate reduction.

b.       If there is no color change, it suggests the reduction of nitrate beyond nitrite, indicating a positive result for nitrate reduction.

Sulfide Indole Motility (SIM) Deeps

1.       Inoculate a SIM deep by stabbing the bacterial isolate into the center of the tube using a sterile inoculating needle.

2.       Incubate the SIM deep at the appropriate temperature for the bacterial isolate for 24-48 hours.

3.       Examine the SIM deep for the following observations:

a.       Blackening of the medium (indicates hydrogen sulfide production).

b.       Formation of a red layer after the addition of Kovac's reagent (indicates indole production).

c.        Diffusion of growth away from the stab line (indicates motility).

4.       Record the results and interpret the metabolic reactions based on the observed changes in the SIM deep.

Citrate Plates

1.       Streak the bacterial isolate onto the surface of a citrate plate using a sterile inoculating loop.

2.       Incubate the citrate plate at the appropriate temperature for the bacterial isolate for 24-48 hours.

3.       Observe the growth of the bacteria on the citrate plate and check for a change in color of the medium.

a.       Blue color, indicating the utilization of citrate as the sole carbon source.

Oxidase Test

1.       Using a sterile inoculating loop, transfer a small amount of the bacterial isolate onto a piece of filter paper or an oxidase test strip.

2.       Add a few drops of oxidase reagent (e.g., N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride) to the filter paper or strip.

3.       Observe for the color development.

a.       Purple or dark blue color within 10-30 seconds, indicating a positive result for the presence of cytochrome c oxidase.

Catalase Test

1.       Using a sterile inoculating loop, transfer a small amount of the bacterial isolate onto a clean glass slide.

2.       Add a few drops of hydrogen peroxide (H2O2) directly to the bacterial colony on the slide.

3.       Observe for the immediate formation of bubbles or effervescence.

a.       Bubbles indicate a positive result for catalase activity.

Coagulase Test

1.       Inoculate a tube of rabbit plasma with the bacterial isolate using a sterile inoculating loop.

2.       Incubate the tube at the appropriate temperature for the bacterial isolate for 4-24 hours.

3.       Observe for changes in the plasma

a.       Formation of a clot within the plasma, indicates a positive result for coagulase activity.

Data Analysis

1.       Compile all the test results and observations for each plate and the identified species.

2.       Compare the growth characteristics and test outcomes between the plates with different acne medications and the control plate.

3.       Analyze the data to draw conclusions about the effects of the acne medications on bacterial growth.

a.       Compare active ingredients, timing, effectiveness, etc.

4.       Write a primary paper using IMRAD formatting for the entirety of this lab.

Review Questions

1.       What is the purpose of inoculating facial bacteria into broth tubes and agar plates?

2.       Why is it important to maintain a clean and sanitized workspace during the lab?

3.       What personal protective equipment (PPE) should be worn while conducting the lab, and why is it necessary?

4.       How can you ensure proper identification and labeling of the sterile broth tubes, TSA plates, and medication tubes?

5.       What is the significance of incubating the inoculated broth tubes and agar plates at specific temperatures?

6.       What growth characteristics of bacterial colonies should be observed and recorded during the culture analysis?

7.       What is the purpose of performing a Gram stain on representative colonies from each plate, and what information can be obtained from this staining procedure?

8.       Why is it important to isolate bacteria from the control plate and the zone of inhibition on each medication plate?

9.       How can Bergey's Manual of Determinative Bacteriology be used in the process of identifying the bacterial species?

10.    What are the indicators used to interpret the results of the Mannitol Fermentation, Bile Tolerance and Esculin Hydrolysis, Hemolysis on Blood Agar, Triple Sugar Iron (TSI) Slants, Methyl Red-Voges-Proskauer (MRVP) Broth, Nitrate Reduction Broth, Sulfide Indole Motility (SIM) Deeps, Citrate Plates, Oxidase Test, and Catalase Test?

11.    How can the data collected from the lab be analyzed to evaluate the effects of acne medications on bacterial growth?

12.    What conclusions can be drawn from the analysis of the data regarding the efficacy of acne medications in inhibiting bacterial growth?

13.    Are there any limitations or potential sources of error in the lab procedures that could impact the interpretation of the results?