The Gram Stain Jacqueline Brooks, MB, SCT, HTL (ASCP) Wally Rosene, PA (ASCP)
9/12/2017 1 NSH Dear Jackie,
Your request to have the workshop Gram Staining for the year 2017 has been approved. NSH staff has added the scheduled session to the NSH Contact Hour Portal so that your attendees can self -report following the meeting.
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Connie Wildeman, MPA Project Coordinator, Education and Events National Society for Histotechnology
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9/12/2017 Connie Wildeman, MPA 2 Project Coordinator, Education and Events National Society for Histotechnology Thank you for a ending Gram Stain. This email is a reminder that to receive a contact hour cer ficate for the mee ng you must report your a endance to NSH through the NSH Contact Hour Portal - ce.nsh.org.
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8/8/2017 4 Objectives
1. The Gram stain is a common stain performed in most laboratories and is often not fully understood. We will review the importance of the gram stain as it pertains to patient care and treatment.
2. The steps in the gram stain will be reviewed with explanation of each step. We hope this will help the attendees understand and be able to troubleshoot common problems with the stain.
3. This presentation will also review bacteria classification to help the attendees better understand the stain
9/12/2017 5 9/12/2017 6 Bacterial classifica on is important, revealing the iden ty of an organism so that its behaviour and likely response to treatment can be predicted. Bacterial structural components Bacterial cell walls are rigid and protect the organism from differences in osmo c tension between the cell and the environment. Gram-posi ve cell walls have a thick pep doglycan layer and a cell membrane, whereas Gram-nega ve cell walls have three layers: inner and outer membranes, and a thinner pep doglycan layer. The mycobacterial cell wall has a high propor on of lipid, including immunoreac ve an gens. Bacterial cell shape can also be used in classifica on. The following cell components are important for classifica on, pathogenicity and therapy. • Capsule: a polysaccharide layer that protects the cell from phagocytosis and desicca on. • Lipopolysaccharide: surface an gens that strongly s mulate inflamma on and protect Gram- nega ve bacteria from complement-mediated lysis. • Fimbriae or pili: specialized thin projec ons that aid adhesion to host cells. Escherichia coli that cause urinary tract infec ons bind to mannose receptors on ureteric epithelial cells by their P fimbriae. Fimbrial an gens are o en immunogenic but vary between strains so that repeated infec ons may occur (e.g. Neisseria gonorrhoeae).
9/12/2017 7 Biological Classifica on
Biological Classifica on Is ed to pa ent treatment plans
9/12/2017 8 Biological Classifica on
• Bacteria-are microscopic living organisms, usually one-celled, that can be found everywhere. They can be dangerous, such as when they cause infec on, or beneficial, as in the process of fermenta on (such as in wine) and that of decomposi on. • Archaea- Any of the unicellular microorganisms that is gene cally dis nct from bacteria and eukaryotes, and o en inhabi ng extreme environmental condi ons • Eukaryota- Organisms whose cells contain a true nucleus • A virus is a small infec ous agent that replicates only inside the living cells of other organisms. Viruses can infect all types of life forms, from animals and plants to microorganisms, including bacteria and archaea
9/12/2017 9 Virus
A virus is a small infec ous agent that replicates only inside the living cells of other organisms. Viruses can infect all types of life forms, from animals and plants to microorganisms, including bacteria and archaea.
9/12/2017 10 Micro-organisms
9/12/2017 11 Bacteria Size
9/12/2017 12 More Classifica on
9/12/2017 13 Gram Stain Classifica on
9/12/2017 14 Gram Posi ve Species
9/12/2017 15 Gram Nega ve Species
9/12/2017 16 Bacterial Classifica on
• Faculta ve means "op onal" or "discre onary" (antonym obligate), used mainly in biology in phrases such as: Faculta ve anaerobe, an organism that can use oxygen but also has anaerobic methods of energy produc on. It can survive in either environment. • Aerobic- (1) Of, pertaining to, having, or requiring the presence of air or free oxygen. (2) (biology) Requiring air or oxygen for life or survival, used especially to refer to aerobic bacteria. (3) (physiology) Pertaining to respira on occurring in the presence of oxygen, as aerobic respira on. • Anaerobic- (1) Not requiring, or capable of occurring, in the absence of air or free oxygen. (2) Caused by, or rela ng to, the lack of molecular oxygen. Supplement. Anaerobic may be used to describe an organism, a cell, a process or a mechanism that can func on without air (i.e. air to generally mean oxygen).
9/12/2017 17 Bacteria
Bacteria are ny, single-celled organisms that are widely distributed in nature. The gene c material of bacteria is not enclosed in a special nuclear membrane, but each bacterial cells is a complete organism that can metabolize, grow, and reproduce. The cell walls that enclose bacteria are primarily composed of a substance called pep doglycan, a mucopolysccharide. Bacteria vary in size form approximately 0.2 to 10 microns in their greatest dimension. They also vary in shape, and this varia on provides a basis for classifica on.
9/12/2017 18 Bacteria
One way of classifying bacteria is by shape. The spherical or ovoid bacteria are classified as cocci and are sub classified according to the way they are arranged. Some cocci occur in pars (diplococci), some occur in grapelike clusters (staphylococci), and others occur in chains (streptococci). Staphylococcus aureus is the agent of toxic shock syndrome and the cause of many life-threatening hospital and locker-room infec ons (e.g. methicillin-resistant Staphylococcus aureus (MRSA).
9/12/2017 19 Bacteria
Other pathogenic cocci are Streptococcus pneumoniae, Neisseria gonorrhoeae, and Neisseria Meningi des. The Rod-shaped bacteria organisms are classified as bacilli. Some of the pathogenic bacilli are Clostridia tetani, Clostridia botulinum, and Bacillus anthracis. Coccobacilli are rod-shaped, but so short and wide that resemble cocci. Examples of coccobacilli are Haemophilus influenza and Chlamydia trachoma s.
9/12/2017 20 Bacteria
Bacteria that are spiral or corkscrew-shaped are classified as spirochetes. Treponema pallidum, the causa ve organism of syphilis, is a very important organism in this group. Borrelia burgdorferi, the organism that causes Lyme disease, is also a spirochete. Silver stains are the primary technique used for the demonstra on of spirochetes.
9/12/2017 21 Bacteria
Ricke siae, chlamydiae, and mycoplasmas are bacteria that do not possess the typical bacterial a ributes. Ricke siae and chlamydiae can reproduce only within a living host cells (ie, obligate intracellular prarsites), and mycoplasmas do not form cell walls. These organisms were originally classified as viruses because of their size and the difficul es encountered in isola ng the organisms.
9/12/2017 22 Bacteria
Chlamydia trachoma s organisms cause lymphogranuloma venereum, trachoma, cervici s, myocardi s, and other diseases in humans, while ricke siae are responsible for such diseases as Rocky Mountain spo ed fever and typhus. Examples of problems caused by myoplasma organisms are pneumonia, s ll birth, and spontaneous abor on.
9/12/2017 23 Bacteria and the Gram Stain
Another way of classifying bacteria is by means of the Gram stain, a stain developed by Chris an Gram in 1884. The stain colors some bacteria deep blue (gram- posi ve) and leaves others either unstained or colored by the counterstain (gram –nega ve). It is now known that the organisms that stain deep blue have a cell wall containing teichoic acid, and those that are unstained or stain red contain lipopolysaccharides. This stain is one of the most basic stains used in microbiology.
9/12/2017 24 Bacteria and the Gram Stain
Salmonella organisms are gram-nega ve bacilli and clostridum botulinum are endospore-forming gram-posi ve rods. Other gram- posi ve bacilli are Clostridium tetani and Corynebacterium diptheriae; other gram-nega ve bacilli are Shigella dysenteriae, Escherichia coli, and Pseudomonas aeruginosa. Neisseria gonorrhoeae and Neisseria meningi des are gram-nega ve cocci, and Streptococcus pneumoniae and Staphylococcus aureus are gram-posi ve.
9/12/2017 25 Bacteria
Many bacteria are pathogenic (disease-producing), and proper iden fica on aids in providing appropriate therapy for the pa ent. Culture, staining, and biochemical characteris cs are all used in the microbiology laboratory to iden fy organisms, while in the histopathology laboratory, iden fica on depends on the shape and staining characteris cs of organisms found in ssue and on the way the ssue has responded to the presence of the organisms. Immunohistochemical methods are also important in the iden fica on of some organisms.
9/12/2017 26 Gram Stain
The “Gram” stain for the demonstra on of bacteria is a popular, widely used technique in both histology and microbiology to delineate gram-posi ve bacteria from gram-nega ve bacteria on smear prepara ons. However, the staining procedures used in the two disciplines vary greatly. In histology, depending upon the procedure used, staining would require from four to 17 steps and almost as many solu ons. The staining me element would vary, again depending upon the procedure used, and staining would take from five to 15 minutes. In contrast, most procedures used in microbiology require from four to seven steps and three to five minutes to complete. Yet techniques used in both disciplines yield the same result. What, then, is the difference between the two? For the most part, the difference found between the two centers mainly around the me element and number of steps involved in the procedure.
9/12/2017 27 Brown-Hopps Modifica on of the Gram Stain
Purpose: Demonstra on of gram-nega ve and gram-posi ve bacteria in ssue Principle: Crystal violet is applied first and then followed by an iodine mordant forming a dye lake. At this point, both gram-nega ve and gram-posi ve organisms are stained. Although both types of bacteria have cell walls composed of pep doglycan and lipoprotein, the cell walls of gram- posi ve bacteria are thick (15-25 nm) than those of the gram-nega ve organisms (8-12 nm).
9/12/2017 28 Brown-Hopps Modifica on of the Gram Stain
Principle: Gram-nega ve bacteria contain irregular layers of lipoprotein and fewer pep doglycan layers, whereas gram-posi ve organisms contain up to 25 layers of pep doglycan in the outer lipoprotein membranes. These differences in the cell wall account for differences in the way that bacteria will decolorize in next procedural step. The large crystal violet-iodine molecular complex cannot easily be washed out of the intact pep doglycan layers of gram-posi ve cells; however, it is easily removed from gram-nega ve bacteria, because alcohol or acetone disrupts the outer lipoprotein layer, and the remaining thin pep doglycan cell wall cannot retain the complex.
9/12/2017 29 Brown-Hopps Modifica on of the Gram Stain
Principle: Gram-posi ve cell walls will retain the crystal violet-iodine complex, unless the cells walls have been damaged or disrupted for some other reason (old or dead organisms). If the cells wall of a normally gram- posi ve organism id damaged, the organism will then stain gram- nega ve. The decoloriza on step is a rela ve one, and sec ons can be over decolorized, removing stain from both gram-nega ve and gram- posi ve organism. A er decoloriza on, a counterstain is applied to color the gram-nega ve organisms.
9/12/2017 30 Brown-Hopps Modifica on of the Gram Stain
Fixa ve: 10% neutral-buffered formalin Equipment: Coplin jars, blo ng paper, Erlenmeyer flasks, graduated cylinders Technique: Cut paraffin sec ons at 4-5 microns Quality Control: Sec ons containing BOTH gram-posi ve and gram-nega ve organisms should be used.
9/12/2017 31 Brown-Hopps Modifica on of the Gram Stain- Reagents Crystal Violet, 1% Solu on: Crystal Violet 5 g Dis lled Water 500 mL Gram Iodine: Iodine 3 g Potassium iodide 6 g Dis lled Water 900 mL Place the iodine and potassium iodide in approximately 150 mL of the water. S r un l completely dissolved, then add the remaining water.
9/12/2017 32 Brown-Hopps Modifica on of the Gram Stain- Reagents Basic Fuchsin Solu on: Basic fuchsin 0.1 g Dis lled Water 100 mL Gallego Solu on: Dis lled water 50 mL Formalin, 37% to 40% 1 mL Glacial ace c acid 0.5 mL Picric Acid-Acetone Solu on: Picric Acid 0.5 g Acetone 1,000 mL
9/12/2017 33 Brown-Hopps Modifica on of the Gram Stain- Procedure
1. Deparaffinize and hydrate sec ons to dis lled water 2. Stain sec ons with crystal violet for 2 minutes 3. Rinse slides in dis lled water 4. Stain slides with gram iodine for 5 minutes 5. Rinse slides in dis lled water to remove excess iodine 6. Blot 1 slide at a me with slightly damp filter paper, and decolorize quickly in acetone. 7. Rinse slides quickly but thoroughly in dis lled water 8. Stain sec ons with working basic fuchsin for 5 minutes 9. Rinse slides in dis lled water 10. Differen ate sec ons with Gallego solu on for 5 minutes 11. Rinse slides in dis lled water and blot sec ons, but do not blot to dryness. 12. Quickly dip slides in acetone 3 mes 13. Quickly dip slides in picric acid-acetone 3 mes 14. Quickly dip slides in acetone 3 mes 15. Pass slides through acetone-xylene mixture (1:2) for 5 quick dips, and then clear with 2 changes of xylene 16. Mount with synthe c resin.
9/12/2017 34 Brown-Hopps Modifica on of the Gram Stain- Results Gram-Posi ve bacteria Blue Gram-Nega ve bacteria Red Background ssue generally Yellow Nuclei Light red
9/12/2017 35 Brown-Hopps Modifica on of the Gram Stain- Technical Notes 1. This modifica on of the Brown and Bren Gram stain is the preferred stain for gram-nega ve organisms and ricke siae, but the original Brown and Brenn procedure is preferred for demonstra ng gram-posi ve bacteria. 2. This method can be useful for screening for the infec ous agents causing ac nomycosis, nocardiosis, coccidiomycosis, blastomycosis, cryptococcosis, aspergillosis, rhinosporidiosis, and amebiasis. 3. The picric acid-acetone decolorizes sec ons be er if the picric acid used to prepare the reagent is nearly anhydrous. Slightly more than the required weight of picric acid can be dehydrated by placing it in a watch glass or glass Copin jar lid in a desiccator overnight. Handle carefully and return any excess to the stock bo le for rehydrate in. Remember that picric acid containing less than 10% water is explosive.
9/12/2017 36 Brown-Hopps Modifica on of the Gram Stain- Technical Notes 4. Sec ons should not be allowed to dry at any stage of the procedure, because drying leads to the forma on of insoluble compounds that are difficult or impossible to decolorize with picric acid-acetone. 5. Brown and Hopps applied the reagents in steps 2,4,8 and 10 to slides that are lying flat; the other steps are done in Coplin jars.
9/12/2017 37 Brown-Hopps Modifica on of the Gram Stain- Technical Notes 6. If picric acid cannot be stocked in your laboratory as a dry reagent, the following steps may be subs tuted: Step a. Immerse slides in 95% ethyl alcohol for 2-3 minutes Step b. Immerse slides in saturated aqueous picric acid (may be obtained commercially) for 13-15 minutes. Step c. Dehydrate and clear as follows: 95% ethyl alcohol, 4 quick dips, 100% alcohol, 2 changes, 8-10 dips each; and xylene, 2 changes, 10 dips each. Step d. Mount with synthe c resin.
9/12/2017 38 Brown-Hopps Modifica on of the Gram Stain- Technical Notes 7. Gram-posi ve organisms most likely will not stain correctly if the pa ent is taking an bio cs, because an bio cs compromise the cell walls.
9/12/2017 39 Gram Stain Sigma-Aldrich (Stain Kit) Intended Use: Gram Stain reagents are intended for use in the delinea on of Gram-Posi ve and Gram-Nega ve organisms in films and ssue. Gram Stain reagents are for “In Vitro Diagnos c Use”. Gram stain is used clinically to delineate two dis nct groups of microorganisms. Those which retain primary dye (crystal violet) are called Gram Posi ve. Those which lose the primary dye during a decoloriz on step are called Gram nega ve. The mechanisms whereby Gram posi ve organisms retain the primary stain are unknown, although the chemistry and structure of cell walls are most certainly involved. Numerous modifica ons of the original Gram method’ have been described. The Sigma-Aldrich procedure is based on the work of Hucker and Coon u lizing a crystal violet-ammonium oxalate solu on that aids in differen a on and is quite stable.
9/12/2017 40 Gram Stain Sigma-Aldrich (Stain Kit)- Reagents Crystal Violet (Cer fied crystal violet, 2.3%, ammonium oxalate, 0.1%, and 20% ethyl alcohol, SD3A. Gram’s Iodine Solu on (Iodine. 0.33%, and potassium iodide, 0.66%) Safranin O Solu on (Cer fied safranin 0.6% in 20% ethyl alcohol, SD3A) Tartrazine Solu on (Tartrazine, 0.25%, and ace c acid, 0.25%)
9/12/2017 41 Gram Stain Sigma-Aldrich (Stain Kit)- Notes Storage and Stability: Store at room temperature (18-26 C). Reagent label bears the expira on da ng. Use once and discard. Prepara on: All reagents are ready to use Precau ons: Normal precau ons exercised in handling laboratory reagents should be followed. Dispose of waste observing all local, state, provincial or na onal regula ons. Refer to Material Safety Data Sheet and product labeling for any updated risk, hazard or safety informa on. Gram Stain Tissue- control slides are paraffin embedded animal ssue containing gram nega ve and gram posi ve bacteria and should be considered poten ally infec ous. 9/12/2017 42 Gram Stain Sigma-Aldrich (Stain Kit) - Procedure
Tissue: Any well fixed paraffin embedded ssue cut at 5 microns 1. Deparaffinize sec ons and hydrate to deionized water 2. Place slides on staining rack and cover sec ons with Crystal Violet Solu on, for 1 minute. 3. Drain off Crystal Violet Solu on and rinse thoroughly in deionized water 4. Mordant in Gram’s Iodine Solu on for 5 minutes 5. Rinse in deionized water and blot sec ons 6. Differen ate in absolute alcohol or acetone 7. Rinse in deionized water 8. Cover slides with Safranin O Solu on 30-60 seconds 9. Rinse in deionized water and blot sec ons. 10. Cover sec ons with Tartrazine Solu on for 5-10 seconds 11. Blot off excess stain 12. Rinse in 2 changes absolute alcohol 13. Clear in xylene and mount
9/12/2017 43 Gram Stain Sigma-Aldrich (Stain Kit) Performance Characteris cs
Gram Posi ve organisms – Purple Gram Nega ve organisms – Red
9/12/2017 44 How do you get gram nega ve bacteria?
• Several species, including Escherichia coli, are common causes of foodborne disease. Vibrio cholerae—the bacteria responsible for cholera—is a waterborne pathogen. Gram-nega ve bacteria can also cause respiratory infec ons, such as certain types of pneumonia, and sexually transmi ed diseases, including gonorrhea.
9/12/2017 45 What are gram posi ve rods?
• Gram-posi ve bacilli—Bacillus anthracis (anthrax) can cause skin infec ons or pneumonia (also a bioterrorism agent); Listeria monocytogenes can cause foodborne illnesses. Gram-nega ve bacilli —Escherichia coli is a common cause of urinary tract infec ons.
9/12/2017 46 What bacteria are gram nega ve cocci?
• Medically relevant gram-nega ve cocci include the four types that cause a sexually transmi ed disease (Neisseria gonorrhoeae), a meningi s (Neisseria meningi dis), and respiratory symptoms (Moraxella catarrhalis, Haemophilus influenzae). Medically relevant gram-nega ve bacilli include a mul tude of species
9/12/2017 47 Why is decoloriza on the most important step in Gram staining?
• The most cri cal phase of the Gram stain procedure is the decoloriza on step, which is based on the ease with which the crystal violet-iodine complex is released from the cell. Over-decolorizing will result in the loss of the primary stain, causing Gram posi ve bacteria to appear Gram nega ve.
9/12/2017 48 What is a mordant in microbiology?
A mordant or dye fixa ve is a substance used to set dyes on fabrics or ssue sec ons by forming a coordina on complex with the dye which then a aches to the fabric or ssue. It may be used for dyeing fabrics, or for intensifying stains in cell or ssue prepara ons.
9/12/2017 49 What an bio cs are used to treat gram nega ve bacteria?
An bio cs that cover Pseudomonas aeruginosa: • Aminoglycosides. • Carbapenems. • Ce azidime (3rd genera on) • Cefepime (4th genera on) • Ce obiprole (5th genera on) • Fluoroquinolones. • Piperacillin/tazobactam. • Ticarcillin/clavulanic acid.
9/12/2017 50 What an bio cs treat gram posi ve rods?
If it has been treated successfully with clindamycin, ciprofloxacin, vancomycin and imipenem. Food poisoning resul ng from this bacillus does not require an bio c treatment. Resistance to penicillin and clindamycin may occur in other Bacillus species, and is common to third-genera on cephalosporins.
9/12/2017 51 Are cocci always gram posi ve?
This shows Gram-posi ve (purple/blue) cocci, and a sca ering of Gram-nega ve (red) cocci. The organism that most commonly infects the skin is staphylococcus aureus, and the younger organisms stain Gram-posi ve, and the older organisms are some mes Gram-nega ve.
9/12/2017 52 Gram Stain
The Gram stain is not an infallible tool for diagnosis, iden fica on, or phylogeny, however. It is of extremely limited use in some instances and has been largely superseded by molecular techniques even in the medical microbiology laboratory. Given that some organisms are Gram- variable (i.e., they may stain either nega ve or posi ve), and that some organisms are not suscep ble to either stain used by the Gram technique. In molecular microbiology laboratory, most iden fica on is done using gene c sequences and other molecular techniques which are far more specific and informa on-rich than differen al staining.
9/12/2017 53 The Gram stain has been used for several purposes that include:
To directly examine specimens submi ed for microbiologic examina on, e.g., body fluid or biopsy when infec on is suspected. It yields results much more quickly than culture, and is especially important when infec on would make an important difference in the pa ent's treatment and prognosis; examples are cerebrospinal fluid for meningi s and synovial fluid for sep c arthri s • To provide preliminary informa on to the clinician regarding presump ve bacterial pathogens • To characterize the type of bacteria growing in culture media (including blood cultures) • And to assess the quality of specimens submi ed for culture (i.e., sputum specimens) to determine whether they are likely to yield clinically useful informa on and whether they should be processed (cultured).
9/12/2017 54 More Pathogenic ?
As a general rule of thumb (which has excep ons), Gram-nega ve bacteria are more pathogenic due to their outer membrane structure. The presence of a capsule will o en increase the virulence of a pathogen. In addi on, Gram-nega ve bacteria have lipopolysaccharide (LPS) in their outer membrane, an endotoxin that increases the severity of inflamma on. This inflamma on may be so severe that sep c shock may occur. Gram-posi ve infec ons are generally less severe because the human body does not contain pep doglycan; in fact, humans produce an enzyme (lysozyme) that a acks the open pep doglycan layer of Gram-posi ve bacteria. Gram- posi ve bacteria are also frequently much more suscep ble to beta-lactam an bio cs, such as penicillin.
9/12/2017 55 More Pathogenic?
Excep ons to the rule include branching and filamentous Gram- posi ve bacteria such as Mycobacterium tuberculosis and other agents of tuberculosis, or Nocardia species, the agents of nocardiosis and some types of ac nomycetoma. These organisms present unique problems in diagnosis and treatment, and special stains such as the Ziehl-Neelsen stain and the Kinyoun stain are used in their laboratory workup.
9/12/2017 56 Clinical Significance of Gram Staining
The Gram stain is a very important preliminary step in the ini al characteriza on and classifica on of bacteria. It is also a key procedure in the iden fica on of bacteria based on staining characteris cs, enabling the bacteria to be examined using a light microscope. The bacteria present in an unstained smear is invisible when viewed using a light microscope. Once stained, the morphology and arrangement of the bacteria may be observed as well. Furthermore, it is also an important step in the screening of infec ous agents in clinical specimens such as direct smears from a pa ent. Although the Gram staining is used for detec on and differen a on of bacteria, other microorganisms, most frequently yeasts and fungi, can be seen on a Gram-stained smear. Like Clostridium organisms, yeasts can appear Gram-posi ve or Gram-nega ve. Yeasts are generally at least 10-20 mes the size of bacteria and hence differen a on from bacteria is not a problem. However, yeasts are the size of CV precipitate, which is occasionally present.
9/12/2017 57 CV precipitate
• This large purple structure can even appear to be budding. CV precipitate can be differen ated from yeasts because:
The precipitate may be present in an area with several other purple ar facts of various size and shape, • The precipitate has a homogenous deep purple color, while the yeast is o en mo led, and • The precipitate may be perfectly round, while Candida species, the yeast most commonly encountered, are generally oval. •
9/12/2017 58 CPT Codes
• 88312- Gram Stain Special stain including interpreta on and report; Group 1 for microorganisms. Report one unit of 88312 for each special stain, on each surgical block, cytologic specimen, or hematologic smear.
9/12/2017 59 The End
9/12/2017 60