The Jacqueline Brooks, MB, SCT, HTL (ASCP) Wally Rosene, PA (ASCP)

9/12/2017 1 NSH Dear Jackie,

Your request to have the workshop Gram 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.

At the Conclusion of the Meeting:

The following items are to be shared with attendees and returned to NSH following the meeting. Missed deadlines may result in NSH revoking the approved contact hours for the session.

1. Session Evaluations:

Evaluation Forms: Each attendee should complete an evaluation form. Forms should be collected at the conclusion of the session and attached to the corresponding Evaluation Summary. Evaluation Summary Form: An evaluation summary form has been provided. It is the responsibility of the Meeting Coordinator to complete the summary calculations for each session and return it with the evaluation forms.

2. Email Text for Post Meeting: As attendees will be responsible for adding contact hours to their records attached is a sample email you can send explaining how it works. Please feel free to edit it as you deem appropriate. Sending this email is not required but recommended.

All paperwork is due to NSH within 30 days of completion of the meeting. Contact hour certificates are immediately available once attendees enter their hours. If NSH does not receive the check in roster or session evaluations access to contact hour certificates will be denied. If you have any questions please feel free to contact our office. Have a great meeting!

Connie Wildeman, MPA Project Coordinator, Education and Events National Society for Histotechnology

NSH has moved! Mailing Address 3545 Ellicott Mills Drive Ellicott City, MD 21043

Payments Address PO Box 75914 Baltimore, MD 21275-5914

Phone | 443.535.4066 [email protected]

Visit NSH's new on-line learning center: learn.nsh.org - Join the conversation: The Block |Twitter |LinkedIn |Facebook

9/12/2017 Connie Wildeman, MPA 2 Project Coordinator, Education and Events National Society for Histotechnology Thank you for aending Gram Stain. This email is a reminder that to receive a contact hour cerficate for the meeng you must report your aendance to NSH through the NSH Contact Hour Portal - ce.nsh.org.

Instrucons for adding your hours to your account are located below. If you have any quesons, please contact the NSH office, 443-535-4060.

Kind regards,

INSERT NAME

Direcons for Subming Your Hours to the NSH Contact Hour Portal 1. Login to NSH Contact Hour Portal – ce.nsh.org (do not use www prior to the web address) a. First Time to the Site? – complete the “Not Yet Registered” form to create your contact hour portal user account. Once you have created a user account you will be asked to complete the user profile form (your name, address etc). To complete the profile and access hours from previous events you will need your NSH Customer ID number in the first step. b. Finding or Geng an NSH Customer ID# - You can log into “My NSH” from nsh.org and find your Customer ID # on the profile page. You can access a short video that explains where to find the number. http://nsh.adobeconnect.com/p3i97kyn5ev/. If you are not a member of NSH or have not aended an event or purchased a book from NSH in the past you may need to create an account through the “My NSH” portal on nsh.org to get your Customer ID#. Here is a short video to explain the process: http://nsh.adobeconnect.com/p1zo0yivh8k/ 2. Once you are logged in select “Session Tracker” from the top navigaon 3. Add your Contact Hours for an event a. Step 1: Year: Select the year in which the event was held. b. Step 2: Event Title: All events approved in a specific year for contact hours by NSH are available to you to in this drop down list. Select the name of the event you aended: Aurora Diagnoscs Webinar Series c. Step 3: Session Title: Select the workshop or webinar you aended: Gram Stain. The number of contact hours awarded for this session is preset and cannot be edited. d. Step 4: Add: Click on Add and your workshop or webinar will appear in your session log listed below. 4. Print your contact hour cerficate a. Click on the box next to each session you would like to appear on your cerficate. b. Make sure you click all of the sessions from one event if you want them to appear on one cerficate. c. If you want all of your hours for a specific year you will need to click on all of the sessions from that year. 9/12/2017 3 Cunningham Pathology,

Your CEU approval documents (Certificate, Approval Letter, and Invoice) have been uploaded to your CEU Vendor Information page. All documents will be available until the listed expiration date. You can access these documents with the following username and password:

Username: cunninghampathology Password: 7LBED6ot0UVi+fO56Wwhu1RfICowiHzBaH7n4xm6nZiIWulbmVNEtTyklLRX2dkeunI5fiao9xzL4 3OF9uoj5Q==

Filter your Recent Activity to approved titles only; your most recent approvals will be at the

bottom of the list. If you login via any other AAPC page, you will need to hover over My AAPC (top right, in the blue bar) and click on Overview under CEU Vendor to see the Recent Activity.

Please give all attendees a copy of the original PDF CEU certificate. This is the only certificate AAPC will accept from a member for CEUs. The PDF is not to be altered, other than adding the name of the recipient and the date of the course.

Please see AAPC CEU Vendor Guidelines for additional information.

Thank you!

AAPC CEU Vendor Department

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 classification to help the attendees better understand the stain

9/12/2017 5 9/12/2017 6 Bacterial classificaon is important, revealing the identy 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 osmoc tension between the cell and the environment. Gram-posive cell walls have a thick pepdoglycan layer and a cell membrane, whereas Gram-negave cell walls have three layers: inner and outer membranes, and a thinner pepdoglycan layer. The mycobacterial cell wall has a high proporon of lipid, including immunoreacve angens. Bacterial cell shape can also be used in classificaon. The following cell components are important for classificaon, pathogenicity and therapy. • Capsule: a polysaccharide layer that protects the cell from phagocytosis and desiccaon. • Lipopolysaccharide: surface angens that strongly smulate inflammaon and protect Gram- negave bacteria from complement-mediated lysis. • Fimbriae or pili: specialized thin projecons that aid adhesion to host cells. Escherichia coli that cause urinary tract infecons bind to mannose receptors on ureteric epithelial cells by their P fimbriae. Fimbrial angens are oen immunogenic but vary between strains so that repeated infecons may occur (e.g. Neisseria gonorrhoeae).

9/12/2017 7 Biological Classificaon

Biological Classificaon Is ed to paent treatment plans

9/12/2017 8 Biological Classificaon

• Bacteria-are microscopic living organisms, usually one-celled, that can be found everywhere. They can be dangerous, such as when they cause infecon, or beneficial, as in the process of fermentaon (such as in wine) and that of decomposion. • Archaea- Any of the unicellular microorganisms that is genecally disnct from bacteria and eukaryotes, and oen inhabing extreme environmental condions • Eukaryota- Organisms whose cells contain a true nucleus • A virus is a small infecous 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 infecous 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 Classificaon

9/12/2017 13 Gram Stain Classificaon

9/12/2017 14 Gram Posive Species

9/12/2017 15 Gram Negave Species

9/12/2017 16 Bacterial Classificaon

• Facultave means "oponal" or "discreonary" (antonym obligate), used mainly in biology in phrases such as: Facultave anaerobe, an organism that can use oxygen but also has anaerobic methods of energy producon. 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 respiraon occurring in the presence of oxygen, as aerobic respiraon. • Anaerobic- (1) Not requiring, or capable of occurring, in the absence of air or free oxygen. (2) Caused by, or relang to, the lack of molecular oxygen. Supplement. Anaerobic may be used to describe an organism, a cell, a process or a mechanism that can funcon 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 genec 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 pepdoglycan, a mucopolysccharide. Bacteria vary in size form approximately 0.2 to 10 microns in their greatest dimension. They also vary in shape, and this variaon provides a basis for classificaon.

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 infecons (e.g. methicillin-resistant Staphylococcus aureus (MRSA).

9/12/2017 19 Bacteria

Other pathogenic cocci are Streptococcus pneumoniae, Neisseria gonorrhoeae, and Neisseria Meningides. 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 trachomas.

9/12/2017 20 Bacteria

Bacteria that are spiral or corkscrew-shaped are classified as spirochetes. Treponema pallidum, the causave 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 demonstraon of spirochetes.

9/12/2017 21 Bacteria

Rickesiae, chlamydiae, and mycoplasmas are bacteria that do not possess the typical bacterial aributes. Rickesiae 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 difficules encountered in isolang the organisms.

9/12/2017 22 Bacteria

Chlamydia trachomas organisms cause lymphogranuloma venereum, trachoma, cervicis, myocardis, and other diseases in humans, while rickesiae are responsible for such diseases as Rocky Mountain spoed fever and typhus. Examples of problems caused by myoplasma organisms are pneumonia, sll birth, and spontaneous aboron.

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 Chrisan Gram in 1884. The stain colors some bacteria deep blue (gram- posive) and leaves others either unstained or colored by the counterstain (gram –negave). 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 .

9/12/2017 24 Bacteria and the Gram Stain

Salmonella organisms are gram-negave bacilli and clostridum botulinum are endospore-forming gram-posive rods. Other gram- posive bacilli are Clostridium tetani and Corynebacterium diptheriae; other gram-negave bacilli are Shigella dysenteriae, Escherichia coli, and Pseudomonas aeruginosa. Neisseria gonorrhoeae and Neisseria meningides are gram-negave cocci, and Streptococcus pneumoniae and Staphylococcus aureus are gram-posive.

9/12/2017 25 Bacteria

Many bacteria are pathogenic (disease-producing), and proper idenficaon aids in providing appropriate therapy for the paent. Culture, staining, and biochemical characteriscs are all used in the microbiology laboratory to idenfy organisms, while in the histopathology laboratory, idenficaon depends on the shape and staining characteriscs 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 idenficaon of some organisms.

9/12/2017 26 Gram Stain

The “Gram” stain for the demonstraon of bacteria is a popular, widely used technique in both histology and microbiology to delineate gram-posive bacteria from gram-negave bacteria on smear preparaons. 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 soluons. 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 Modificaon of the Gram Stain

Purpose: Demonstraon of gram-negave and gram-posive bacteria in ssue Principle: is applied first and then followed by an iodine mordant forming a dye lake. At this point, both gram-negave and gram-posive organisms are stained. Although both types of bacteria have cell walls composed of pepdoglycan and lipoprotein, the cell walls of gram- posive bacteria are thick (15-25 nm) than those of the gram-negave organisms (8-12 nm).

9/12/2017 28 Brown-Hopps Modificaon of the Gram Stain

Principle: Gram-negave bacteria contain irregular layers of lipoprotein and fewer pepdoglycan layers, whereas gram-posive organisms contain up to 25 layers of pepdoglycan 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 pepdoglycan layers of gram-posive cells; however, it is easily removed from gram-negave bacteria, because alcohol or acetone disrupts the outer lipoprotein layer, and the remaining thin pepdoglycan cell wall cannot retain the complex.

9/12/2017 29 Brown-Hopps Modificaon of the Gram Stain

Principle: Gram-posive 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- posive organism id damaged, the organism will then stain gram- negave. The decolorizaon step is a relave one, and secons can be over decolorized, removing stain from both gram-negave and gram- posive organism. Aer decolorizaon, a counterstain is applied to color the gram-negave organisms.

9/12/2017 30 Brown-Hopps Modificaon of the Gram Stain

Fixave: 10% neutral-buffered formalin Equipment: Coplin jars, blong paper, Erlenmeyer flasks, graduated cylinders Technique: Cut paraffin secons at 4-5 microns Quality Control: Secons containing BOTH gram-posive and gram-negave organisms should be used.

9/12/2017 31 Brown-Hopps Modificaon of the Gram Stain- Reagents Crystal Violet, 1% Soluon: Crystal Violet 5 g Dislled Water 500 mL Gram Iodine: Iodine 3 g Potassium iodide 6 g Dislled Water 900 mL Place the iodine and potassium iodide in approximately 150 mL of the water. Sr unl completely dissolved, then add the remaining water.

9/12/2017 32 Brown-Hopps Modificaon of the Gram Stain- Reagents Basic Fuchsin Soluon: Basic fuchsin 0.1 g Dislled Water 100 mL Gallego Soluon: Dislled water 50 mL Formalin, 37% to 40% 1 mL Glacial acec acid 0.5 mL Picric Acid-Acetone Soluon: Picric Acid 0.5 g Acetone 1,000 mL

9/12/2017 33 Brown-Hopps Modificaon of the Gram Stain- Procedure

1. Deparaffinize and hydrate secons to dislled water 2. Stain secons with crystal violet for 2 minutes 3. Rinse slides in dislled water 4. Stain slides with gram iodine for 5 minutes 5. Rinse slides in dislled 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 dislled water 8. Stain secons with working basic fuchsin for 5 minutes 9. Rinse slides in dislled water 10. Differenate secons with Gallego soluon for 5 minutes 11. Rinse slides in dislled water and blot secons, 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 synthec resin.

9/12/2017 34 Brown-Hopps Modificaon of the Gram Stain- Results Gram-Posive bacteria Blue Gram-Negave bacteria Red Background ssue generally Yellow Nuclei Light red

9/12/2017 35 Brown-Hopps Modificaon of the Gram Stain- Technical Notes 1. This modificaon of the Brown and Bren Gram stain is the preferred stain for gram-negave organisms and rickesiae, but the original Brown and Brenn procedure is preferred for demonstrang gram-posive bacteria. 2. This method can be useful for screening for the infecous agents causing acnomycosis, nocardiosis, coccidiomycosis, blastomycosis, cryptococcosis, aspergillosis, rhinosporidiosis, and amebiasis. 3. The picric acid-acetone decolorizes secons beer 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 bole for rehydrate in. Remember that picric acid containing less than 10% water is explosive.

9/12/2017 36 Brown-Hopps Modificaon of the Gram Stain- Technical Notes 4. Secons should not be allowed to dry at any stage of the procedure, because drying leads to the formaon 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 Modificaon 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 substuted: 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 synthec resin.

9/12/2017 38 Brown-Hopps Modificaon of the Gram Stain- Technical Notes 7. Gram-posive organisms most likely will not stain correctly if the paent is taking anbiocs, because anbiocs 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 delineaon of Gram-Posive and Gram-Negave organisms in films and ssue. Gram Stain reagents are for “In Vitro Diagnosc Use”. Gram stain is used clinically to delineate two disnct groups of microorganisms. Those which retain primary dye (crystal violet) are called Gram Posive. Those which lose the primary dye during a decolorizon step are called Gram negave. The mechanisms whereby Gram posive organisms retain the primary stain are unknown, although the chemistry and structure of cell walls are most certainly involved. Numerous modificaons of the original Gram method’ have been described. The Sigma-Aldrich procedure is based on the work of Hucker and Coon ulizing a crystal violet-ammonium oxalate soluon that aids in differenaon and is quite stable.

9/12/2017 40 Gram Stain Sigma-Aldrich (Stain Kit)- Reagents Crystal Violet (Cerfied crystal violet, 2.3%, ammonium oxalate, 0.1%, and 20% ethyl alcohol, SD3A. Gram’s Iodine Soluon (Iodine. 0.33%, and potassium iodide, 0.66%) O Soluon (Cerfied safranin 0.6% in 20% ethyl alcohol, SD3A) Tartrazine Soluon (Tartrazine, 0.25%, and acec 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 expiraon dang. Use once and discard. Preparaon: All reagents are ready to use Precauons: Normal precauons exercised in handling laboratory reagents should be followed. Dispose of waste observing all local, state, provincial or naonal regulaons. Refer to Material Safety Data Sheet and product labeling for any updated risk, hazard or safety informaon. Gram Stain Tissue- control slides are paraffin embedded animal ssue containing gram negave and gram posive bacteria and should be considered potenally infecous. 9/12/2017 42 Gram Stain Sigma-Aldrich (Stain Kit) - Procedure

Tissue: Any well fixed paraffin embedded ssue cut at 5 microns 1. Deparaffinize secons and hydrate to deionized water 2. Place slides on staining rack and cover secons with Crystal Violet Soluon, for 1 minute. 3. Drain off Crystal Violet Soluon and rinse thoroughly in deionized water 4. Mordant in Gram’s Iodine Soluon for 5 minutes 5. Rinse in deionized water and blot secons 6. Differenate in absolute alcohol or acetone 7. Rinse in deionized water 8. Cover slides with Safranin O Soluon 30-60 seconds 9. Rinse in deionized water and blot secons. 10. Cover secons with Tartrazine Soluon 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 Characteriscs

Gram Posive organisms – Purple Gram Negave organisms – Red

9/12/2017 44 How do you get gram negave bacteria?

• Several species, including Escherichia coli, are common causes of foodborne disease. Vibrio cholerae—the bacteria responsible for cholera—is a waterborne pathogen. Gram-negave bacteria can also cause respiratory infecons, such as certain types of pneumonia, and sexually transmied diseases, including gonorrhea.

9/12/2017 45 What are gram posive rods?

• Gram-posive bacilli—Bacillus anthracis (anthrax) can cause skin infecons or pneumonia (also a bioterrorism agent); Listeria monocytogenes can cause foodborne illnesses. Gram-negave bacilli —Escherichia coli is a common cause of urinary tract infecons.

9/12/2017 46 What bacteria are gram negave cocci?

• Medically relevant gram-negave cocci include the four types that cause a sexually transmied disease (Neisseria gonorrhoeae), a meningis (Neisseria meningidis), and respiratory symptoms (Moraxella catarrhalis, Haemophilus influenzae). Medically relevant gram-negave bacilli include a multude of species

9/12/2017 47 Why is decolorizaon the most important step in Gram staining?

• The most crical phase of the Gram stain procedure is the decolorizaon 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 posive bacteria to appear Gram negave.

9/12/2017 48 What is a mordant in microbiology?

A mordant or dye fixave is a substance used to set dyes on fabrics or ssue secons by forming a coordinaon complex with the dye which then aaches to the fabric or ssue. It may be used for dyeing fabrics, or for intensifying stains in cell or ssue preparaons.

9/12/2017 49 What anbiocs are used to treat gram negave bacteria?

Anbiocs that cover Pseudomonas aeruginosa: • Aminoglycosides. • Carbapenems. • Ceazidime (3rd generaon) • Cefepime (4th generaon) • Ceobiprole (5th generaon) • Fluoroquinolones. • Piperacillin/tazobactam. • Ticarcillin/clavulanic acid.

9/12/2017 50 What anbiocs treat gram posive rods?

If it has been treated successfully with clindamycin, ciprofloxacin, vancomycin and imipenem. Food poisoning resulng from this bacillus does not require anbioc treatment. Resistance to penicillin and clindamycin may occur in other Bacillus species, and is common to third-generaon cephalosporins.

9/12/2017 51 Are cocci always gram posive?

This shows Gram-posive (purple/blue) cocci, and a scaering of Gram-negave (red) cocci. The organism that most commonly infects the skin is staphylococcus aureus, and the younger organisms stain Gram-posive, and the older organisms are somemes Gram-negave.

9/12/2017 52 Gram Stain

The Gram stain is not an infallible tool for diagnosis, idenficaon, 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 negave or posive), and that some organisms are not suscepble to either stain used by the Gram technique. In molecular microbiology laboratory, most idenficaon is done using genec sequences and other molecular techniques which are far more specific and informaon-rich than differenal staining.

9/12/2017 53 The Gram stain has been used for several purposes that include:

To directly examine specimens submied for microbiologic examinaon, e.g., body fluid or biopsy when infecon is suspected. It yields results much more quickly than culture, and is especially important when infecon would make an important difference in the paent's treatment and prognosis; examples are cerebrospinal fluid for meningis and synovial fluid for sepc arthris • To provide preliminary informaon to the clinician regarding presumpve bacterial pathogens • To characterize the type of bacteria growing in culture media (including blood cultures) • And to assess the quality of specimens submied for culture (i.e., sputum specimens) to determine whether they are likely to yield clinically useful informaon and whether they should be processed (cultured).

9/12/2017 54 More Pathogenic ?

As a general rule of thumb (which has excepons), Gram-negave bacteria are more pathogenic due to their outer membrane structure. The presence of a capsule will oen increase the virulence of a pathogen. In addion, Gram-negave bacteria have lipopolysaccharide (LPS) in their outer membrane, an endotoxin that increases the severity of inflammaon. This inflammaon may be so severe that sepc shock may occur. Gram-posive infecons are generally less severe because the human body does not contain pepdoglycan; in fact, humans produce an enzyme (lysozyme) that aacks the open pepdoglycan layer of Gram-posive bacteria. Gram- posive bacteria are also frequently much more suscepble to beta-lactam anbiocs, such as penicillin.

9/12/2017 55 More Pathogenic?

Excepons to the rule include branching and filamentous Gram- posive bacteria such as Mycobacterium and other agents of tuberculosis, or Nocardia species, the agents of nocardiosis and some types of acnomycetoma. 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 inial characterizaon and classificaon of bacteria. It is also a key procedure in the idenficaon of bacteria based on staining characteriscs, 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 infecous agents in clinical specimens such as direct smears from a paent. Although the Gram staining is used for detecon and differenaon of bacteria, other microorganisms, most frequently yeasts and fungi, can be seen on a Gram-stained smear. Like Clostridium organisms, yeasts can appear Gram-posive or Gram-negave. Yeasts are generally at least 10-20 mes the size of bacteria and hence differenaon 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 differenated from yeasts because:

The precipitate may be present in an area with several other purple arfacts of various size and shape, • The precipitate has a homogenous deep purple color, while the yeast is oen moled, 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 interpretaon 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