•The century old Gram stain remains the mainstay of rapid diagnosis. Bio 139 Labs #9 and #10: Gram's Stain & KOH test Gram Stain (Lab #9) • One of the most utilized procedures in all of microbiology • Differential stain --Allows you to distinguish 2 groups of bacteria --Used for morphology Differentiates cells by chemical composition of cell wall • Gram positive vs. A Gram stain is a lab test used to detect bacteria or fungi in a sample taken from the site of a Lab Tests Online is an award-winning patient education website offering information on laboratory tests. Gram Staining Now that the slide has been heat fixed, you may now begin to stain the organism. • House color prints in unbuffered, unprinted, neutral pH,. A survey of the 12 medical residents on the wards that month revealed that they routinely read ECG. Blood cultures on admission grew extended spectrum β-lactamase-positive Klebsiella pneumoniae and ertapenem was prescribed. Individual bacteria observed using a microscope, descriptions included, focus on a single bacterium (research)__3__2__1__0 Gram stain procedure attempted, student was able to stain and identify types of bacteria __3__2__1__0 Overall Presentation - report shows attention to detail, provides evidence from the lab, organized __3__2__1__0. In particular, the presence of disease-causing. Reagents of Gram Stain Because the Gram stain reaction is more widely used and studied than any other stain, it is also the most open to variation in protocol. If methylene blue stain is applied to a sample, a healthy cell with turn the stain colorless. Bacteria can be classified as gram-positive and gram-negative depending on their cellular wall. Gram staining: differentiates bacteria into two types Gram positive and Gram negative bacteria. Gram positive bacteria will typically have a stronger affinity for crystal violet on. Real-time poly-merase chain reaction (RT-PCR) of CSF has been. The cultures's response to differential staining - Gram-stain quality (Gram-positive or Gram-negative) and if or not the organisms present are acid-fast. Then submit your finished lab report as a Microsoft Word document. The cell wall can stain either positive or negative, depending on its chemistry. The only work limit is for the Abstract section (see below). Lab Report Expectations. exercise 5 Gram Staining Purpose 43 LABORATORY REPORT Escherichia coli Name Date Lab Section Bacillus subtilis The smallest? Data —Use colora Staphylococcus epidermidis Sketch a few bacteria (1000 X). Acid fast cell walls are so durable that the stain (carbol fuschin) must be driven into the cells with heat. Gram Stain Isolation of Cultures Endospore Stain Differential Media and Enzymatic Assays Enzymatic Assays. Alkaline Phosphatase Live Stain is a stem cell imaging product that allows users to differentially stain pluripotent stem cells (PSCs). doc November 7,,, 2001 Mollleculll ar Celll lll Physiiiolll ogy Protocolll Staining procedure 1. The Gram stain procedure has been basically unchanged since it was first developed in 1884. Lets Summarise Table #1 and #2. Most bacteria are classified into two broad categories: Gram positive and Gram negative. gram's stain and culture of the sputum (Chinnnusamy et al. These are the sources and citations used to research Microbiology gram staining lab report. No results found for your search criteria. Glass slides 3. Quickly memorize the terms, phrases and much more. Read through the case study and assign the Gram Stain status. Bacteria that retain the initial crystal violet stain (purple) are said to be ''Gram-positive,'' whereas those that are decolorized and stain red with carbol fuchsin (or safranin) are said to be ''Gram-negative. The gram stain for the Unknown A bacterium showed results for gram positive rods. U/24 h : units per 24 hours. The Gram stain should almost always be done as the first step in diagnosis of a bacteria infection. The purpose of this study was to evaluate the usefulness of sputum Gram stain in etiological diagnosis and pathogen-targeted antibiotic treatment of CAP and HCAP. gram crystal violet, 16 oz. On the first day of class, you received a handout outlining the laboratory rules. It is maintained by IT Services and the Office of the CIO. o Positive antigen test for N. This stain will either stain the cells purple (for positive) or pink (for negative). The alcohol dehydrates Gram_positive Bacteria, which have very thick cell walls consisting of several layers of peptidoglycan. Purpose: is to determine the Gram stain of your bacterial sample. The alternative staining used is Methylene Blue solution (0,5 %, 0,7%,. To discolor, soil, or spot: The spilled juice stained the carpet. It is the type of "Differential staining" which makes the use of more than one stains to differentiate the bacteria. Today, there are many types of culture media available commercially but the two major types are agar plates and nutrient broth, used depending on the type of cell to be cultured. The testing range for Ampicillin is 0. Processing Your Order. 16 Reporting preliminary blood culture results. Case Classification Suspect: Clinical purpura fulminans in the absence of a positive blood culture; OR Gram-negative diplococci, not yet identified, isolated from a normally sterile body site. Gram Stain Purpose, Procedure and Preparation. Learn gram stain lab 7 with free interactive flashcards. • Place original slides, negatives, transparencies, and prints in individual sleeves to protect them. Although simple stains are useful, they do not reveal details about the bacteria other than morphology and arrangement. By forming spores, bacteria can survive in hostile conditions. We use this new test in our lab to complement the results of the Gram test and not as a replacement for the commonly used Gram stain. Your Name:. For samples from other sites, please order the appropriate culture (which includes Gram stain). Unknown Lab Report: Unknown "C" Discussion/Results. [I1e] Moraxella/Neisseria species: gram-negative diplococci with kidney-bean or biscuit-shaped ends (arrow). Brown, MD, FCAP. Sharkey, MD, FCAP University of Texas Health Science Center, San Antonio, TX. VETERINARY DIAGNOSTIC CENTER General Submission Form Office Use Only FRM-VDC-014 1. AFB laboratory tests detect the bacteria in a person's sample and help identify an infection. Download with Facebook. Gram's Iodine Solution 6. Processing Your Order. False Gram Negative Staining. Gram in 1884 it remains an important and useful technique to this day. Since the unknown bacteria was a gram negative rod, a gelatin test was performed and the organism was inoculated into a. Sputum and ETSAs with ≤10 SECs per LPF were considered to be adequate for Gram stain report and for culture, whereas those with >10 SECs per LPF were rejected. Gram - Stains and differentiates both Gram (+) and Gram (-) bacteria. Colonization with gram negative bacilli and other nosocomial pathogens are common in patients with endotracheal tubes. The final assignment asked the students to complete a lab report in which they discussed Gram staining procedure and the general color change they observed macroscopically. The reason a Gram Stain was attempted was to identify the type of bacterium and determine whether or not it truly had been isolated (McDonald et al. It has to be one of the most repeated procedures done in any lab. The procedure. Acid-fast Stain A. The Gram stain is a type of differential stain that allows a (micro)biologist to. Organisms were seen in the Gram stain in 102/116 samples. The Gram staining technique is the most important and widely used microbiological differential staining technique. In some rare cases, Gram stain and KOH test results differ. Read through the case study and assign the Gram Stain status. Module: The Gram Stain Procedure - click “start lab” From a liquid culture, take a loopful of bacteria emulsify it in a small drop of water or saline on the slide. Only 1 in 10 will see an autopsy. Alt 5 was given as a substitute for a gram-negative bacterium, due to no growth in original test tube (#122). 77-79) sections of the Biology Student Handbook • Answer the following questions in your lab notebook: o How can we test the accuracy of a micropipette?. Gram was searching for a method that would allow visualization of cocci in tissue sections of lungs of. Tellurite agar, therefore, is used to select for Gram-positive organisms, and nutrient agar supplemented with penicillin can be used to select for Gram-negative organisms. The initial impression was that of actinomycetes although the gram-negative staining was contradictory. This is the major difference. Prepare a Gram stain from the NA slant. Methods: CSF specimens received at a tertiary microbiology laboratory between January 1, 2013, and December 31, 2013, were included. Bacterial cultures 2. 73% and Nihan Ziyade et al- 78. Product literature. Occurrence of a phosphoric ester in certain bacteria: its relation to gram staining and penicillin sensitivity. Clinical. Gram Staining: A. diphtheriae - Gram stain (Gram. Diagnostic evaluation of liver tissue is largely based on a thorough examination of sections stained with hematoxylin and eosin (H&E). Gram Postive Bacteria - These are bacteria that have a thick peptidoglycan layer which the stain (crystal violet-iodine complex) is attracted to. pneumoniae, which was detectable with Gram staining. Plates (5% sheep blood, Columbia with colistin and nalidixic acid, MacConkey and chocolate agars) were incubated for 48 h in 5% CO 2 and read after 24 h and 48 h. The presence of a faint smaller shape throughout the slide was also noticed, and thought to be spores. Mordants are substance that improves the staining property of the dye. 1 Introduction. First described by Danish pathologist Christian. It is an alkaline substance staining that can staining the spore bacteria. The name comes from the Danish bacteriologist Hans Christian Gram, who developed the technique. Gram Stain Category Africa America American History Ancient Art Asia Biographies Book Reports Business Creative Writing Dance Economics English Europe History Humanities Literature Medicine Middle East Miscellaneous Music and Movies Philosophy Poetry & Poets Psychology Religion Science Shakespeare Social Issues Speeches Sports Technology TV. Anna Guanipa Period 1 - Microbiology Gram Stain Lab Report Gram Stain Introduction A gram stain is a systematic procedure done to identify different types of bacteria. –Inexpensive to. Unless it is a gram stain of a pure culture, there normally should be material staining both colors somewhere on the slide. Gram stain protocols. Study Flashcards On Microbiology: Lab Exercise 5-Gram Staining at Cram. Box 82646 Lincoln, NE 68501-2646. (Kits are available commercially, or formulas for reagents are listed in Appendix B. (1, 2, 3, and 4). The technique is based on dilution of the sample in a medium, followed by filtration through a porous polysulfone membrane with a pore size retaining and concentrating bacteria from the sample. Show more Show less. Created Date: 9/16/2005 1:11:26 PM. Note: When the tap water is not clean, wash the smear with filtered water or clean boiled rainwater. Department of Health and Human Services, Public Health Service, Centers for Disease Control , 1984 - Gram's stain - 70 pages. Notes from Microbiology. Streak a lawn of Streptococcus pneumoniae onto one half and Alpha strep viridans onto the other. Learning to use the microscope to view what type of bacteria is contaminating the. Smith, Marise A. A gram stain was taken from a sample of the bacteria growing on the nutrient agar. Promote rapid delivery of specimens to the laboratory (goal is delivery in lab within 24 hrs from collection) Use fluorescent acid-fast staining and microscopic examination (goal reporting AFB smear result within 24 hrs from receipt of specimen in lab) Use rapid broth system for primary culture detection of AFB. It is one of the differential stains that are used to characterize bacteria in one of two groups: either gram positive bacteria or gram negative bacteria. It is also known as Gram staining or Gram's method. Yunusa, et al. Skillful Microbiology Gram Stain Flow Chart Flow Chart For Unknown Bacteria Identification Clinical Microbiology Flow Charts Gram Negative Rods Chart - Web 2020 Best Site Medical Lab Technician Gram Negative Bacteria Student Info Medical Laboratory Science Custom Website Design Nursing Students Medical Students Science Projects New Tricks. These Gram-positive thermophilic organisms ferment the sugar lactose to lactic acid via glycolysis. Staining Type # 3. The technologist in the routine microbiology section will examine the direct smear for bacteria and yeast but may. coli with a MIC <= 8 is interpreted as susceptible, an MIC of 16 is intermediate, and >= 32 is resistant. The Gram stain is an example of a microbiology test that requires interpretation by the medical laboratory technologist. 9 Advantages and Disadvantages of Gram Staining Jan 14, 2017 Oct 26, 2016 by Editor in Chief Gram staining, which is also referred to as the Gram’s method, is a scientific technique of staining that is used to differentiate the species of bacteria into 2 main groups, namely the gram-positive and the gram-negative bacteria. Uncategorized. inhibit the growth of Gram-negative bacteria. Therefore, a retest of the bacterium was performed, and finally, V vulnificus was confirmed by 16S rRNA. If > moderate pathogen not seen on original Gram stain, review Gram stain. Most bacteria have no colour, so they generate little contrast in the microscope field. The procedure is based on the ability of. Positive Gram stain report indicates type of microorganism & their morphology based on the type of cells seen. Simple and Gram Staining - Free download as Word Doc (. The oxidase test was performed on unknown #122 gram positive, showed to be positive. Bio 1 River Ecology Lab Report Ecology is the study of relationships between organisms and their environment which includes both physical and biological factors. Bacteria that are non-responsive to this technique are known as gram insensitive. Bacteria Sampling (formerly Disposable Lab Equipment) should be followed by Gram Staining, then Using a Microscope This work was supported by USDA CSREES and USDA National Institute of Food and Agriculture under two Higher Education Challenge Grant projects: 2008-38411-19055 and 2011-38411-30625. Spore formation is an activity performed by bacteria, not for reproductive purposes, but for survival purposes. This test is done to determine whether the bacteria is gram positive (+) or gram negative ( - ). It has to be one of the most repeated procedures done in any lab. Difference Between Gram Stain and Acid Fast www. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle. Next, cover the slide with Gram’s Iodine and let sit for a full minute before rinsing with water for another 5 seconds. It was developed by Danish microbiologist Hans Christian Gram in 1884 as an effective method to distinguish between bacteria with different types of cell walls, and even today it remains one of the most frequently used staining techniques. Type in “prglucose” (no spaces) and click “okay”. An nigrosin-eosin stain is commonly used because it is effective, simple and, in addition to allowing sperm to be readily visualized, it is a so-called "live-dead" stain, allowing one to assess membrane integrity at the same time as morphology. The stain will be examined for the absence or presence of inflammatory cells, and specific bacterial morphotypes and staining patterns are noted. Flagella Staining: West and Difco's SpotTest Methods PART THREE Basic Laboratory and Culture Techniques 13. It is the type of "Differential staining" which makes the use of more than one stains to differentiate the bacteria. First described by Danish pathologist Christian. Cover with CRYSTAL VIOLET for 20 seconds. The Gram Stain and Differential Staining. Perform a successful capsule stain to distinguish capsular material from the bacterial cell. Learn gram stain lab 7 with free interactive flashcards. Learn this handout well; it will serve you greatly during the semester. A Gram stain can also serve to assess the quality of a clinical specimen. Christian Gram in 1884 introduces Gram staining technique and classified bacteria on its Gram characters which might be Gram-negative or Gram-positive. Colonial morphology and endospore stain to confirm Bacillus cereus. LABORATORY IDENTIFICATION OF VIBRIO CHOLERAE. The extra time and care taken during the field season will be rewarded later when the smears must be scanned, and parasites identified and counted. MODULE Morphology and Classification of Bacteria Microbiology 6 Notes Differential Stains These stains impart different colours to different bacteria or bacterial structures, the two most widely used differential stains are the Gram stain and Acid fast stain. Gram stain or Gram staining, also called Gram's method, is a method of staining used to distinguish and classify bacterial species into two large groups: gram-positive bacteria and gram-negative bacteria. Experiment 2. Bright Belinda Afra. Presence of pus cells Gram Positive Cocci. GRAM STAIN TECHNIQUE Page 3 of 6 Gram Kleuring. The gram stain is often the basis of the report. The Gram stain may indicate the need for additional media or a change in the incubation conditions. PEA is a selective media which functions by allowing. Information technology resources, news, and service information at MSU. Eyram Ganu Gram Staining Introduction In this lab experiment, we were introduced to the technique of gram. Role of pathogens which are isolated from non acceptable sputum samples causing LRTIs is uncertain. CSF Gram staining and real-time PCR are theoretically less affected by. Perform Experiment 26: Gram-positive Cocci, Lab. Mordants are substance that improves the staining property of the dye. The Gram stain and final culture results are shown in figure 1 and table 3. No further tests were run on the growth on Plate 9 because it was not an isolated colony of bacteria. AFB laboratory tests detect the bacteria in a person's sample and help identify an infection. This lab helps students develop an understanding of the biochemical and molecular differences in bacteria and introduces the concept of identifying species based on characeristic gene sequences. A sputum Gram’s stain is a quick, low-risk laboratory test. It was developed by Hans Christian Gram; a Danish physician, in 1884. SPECIMEN SOURCE 6. Click here to print a PDF copy of this lab report. Role of pathogens which are isolated from non acceptable sputum samples causing LRTIs is uncertain. Transfer and Isolation Techniques. Simple and Gram Staining of Bacteria And Aseptic transfer of microorganisms and proper handling of bacteria. MICROBIAL MORPHOLOGY, DIFFERENTIAL STAINS. MCB 3020L Lab Experiment 2 The Gram Stain A one lab session experiment In most microbiological staining procedures, the bacteria are first fixed to the slide by the heat fixed smear. After determining the Gram reaction, specific biochemical tests were performed. that most bacteria are transparent and cannot be seen through the microscope. It is used to distinguish between gram-positive and gram-negative bacteria, which have distinct and consistent differences in their cell walls. No results found for your search criteria. Also look up all of these stains in your Leboffe and Pierce book. The isolated bacteria are stained by various methods depending upon the bacteria in focus. The different response of the two groups to the Gram stain is based on fundamental differences in cell wall structure and composition. Since the unknown bacteria was a gram negative rod, a gelatin test was performed and the. Can be identified by Gram staining (gram positive or gram negative) Gram Staining. Laboratory Methods for the Diagnosis of Vibrio cholerae Centers for Disease Control and Prevention VI. 88 Date: Title: Exercise #2 Optochin Disc. Since unknown G. A poor slide is a torment. This should be a thin, not milky, suspension or it will not stain properly. Acid-fast Stain. Microbiology Lab Report- Gram Staining, Simple Staining, Negative Staining. module_instructions. Mycobacteria are called acid-fast bacilli because they are a group of rod-shaped bacteria (bacilli) that can be seen under the microscope following a staining procedure where the bacteria retain the color of the stain after an acid wash (acid-fast). The technologist in the routine microbiology section will examine the direct smear for bacteria and yeast but may. Interval (Microscopy) GRAM STAIN. The terms positive and negative have nothing to do with electrical charge, but simply designate two distinct morphological groups of bacteria. Gram staining indicated the presence of long pinkish rods with circular purple spots inside the cells. Thus, various staining methods are developed to facilitate bacterial detection. Bacteria can form endospores in approximately 6 to 8. Then submit your finished lab report as a Microsoft Word document. Sputum and ETSAs with ≤10 SECs per LPF were considered to be adequate for Gram stain report and for culture, whereas those with >10 SECs per LPF were rejected. Gram Stain for Diagnosing BV ‹Based on the quantity of 3 morphotypes -Lactobacilli, Gram variable rods, and curved rods ‹Sensitivity 86%-89% and specificity 94%-96% compared to Amsel criteria ‹Will be done at Central Lab in Pittsburgh. Although the Gram stain is the most widely used stain in microbiology laboratories, it is not routinely recommended for the laboratory diagnosis of cryptococcal meningitis. Stir the end of the toothpick in the stain and throw the toothpick away. FLOWCHART (Removed Due to Formatting Issues with Blog) UNKNOWN #120 "B" DISCUSSION/CONCLUSION: Unknown #120 "A"- Escherichia coli The test results of the biochemical tests help to determine the final identification for the bacterium, Escherichia coli. Michelle Landreville: Ā: Writing Ree,Pe, FeQ, Pa. Gram stains are usually scanned for the presence or absence. From a liquid culture, take a loopful of bacteria emulsify it in a small drop of water or saline on the slide. Uncategorized. U/6 h : units per 6 hours. Streak a lawn of Streptococcus pneumoniae onto one half and Alpha strep viridans onto the other. Gram-negative bacteria are a group of bacteria that do not retain the crystal violet stain (when rinsed with alcohol) used in the Gram staining method of bacterial differentiation, [1] making positive identification possible. Different stains are used to perform this process; crystal violet, Gram’s iodine, ethyl alcohol, and sapphire stain. Gram Staining Formal Lab Report Miranda King Intro Microbiology Dr. • House color prints in unbuffered, unprinted, neutral pH,. 46, df = 8, p <. : Laboratory perspective of Gram staining and its signi fi cance in investigations of infectious diseases 173 Sub-Saharan African Journal of Medicine /Vol 1/Issue 4/Oct-Dec 2014 d. The Gram stain is the most widely used staining procedure in bacteriology. Pour off the Gram's iodine. The Gram stain is a popular microbiology technique used to differentiate between two groups of bacteria based on cell wall composition. 50 Gram-positive bacteria appear purple in color and gram-negative bacteria appear pink. Isolation 4. Bacteria can be classified as gram-positive and gram-negative depending on their cellular wall. The interactive transcript could not be loaded. Optional: Supplementary Gram Program. Leave it for one minute then wash it with tap water on sink using a holder to remove excess stain. It is one of the differential stains that are used to characterize bacteria in one of two groups: either gram positive bacteria or gram negative bacteria. the acid-fast stain for Mycobacterium tuberculosis , fungal culture, and rapid identification tests [tests for bacterial and fungal antigens]) may be performed routinely. Gram stain • Han Christian Gram: 1884 • Method of classifying bacteria into 2 large groups: Gram positive (+) and Gram negative (-) • Differentiates bacteria by the chemical and physical properties of their cell walls • Helpful in guiding initial empiric therapy • Reported to physicians ASAP 11 11 Gram positive: thick cell wall. coli have an additional outer membrane that is composed of. Learn gram stain lab 7 with free interactive flashcards. The extra time and care taken during the field season will be rewarded later when the smears must be scanned, and parasites identified and counted. Depending on the bacteria take up and retain the primary stain; crystal violet during the gram staining, there are two types of bacteria namely gram positive and gram negative. Transport to the Bacteriology Laboratory immediately to prevent loss of specimen due to drying. The Gram stain was first used in 1884 by Hans Christian Gram (Gram,1884). There was a 100% correlation between the 2 methods. [PMC free article] [Google Scholar] MITCHELL P, MOYLE J. the colonization of the unknown bacteria (Lammert 2007). diphtheriae - Neisser stain (violet storage cells) C. Laboratory Report 15 15 Gram Staining A. Any basic dye such as methylene blue, safranin, or crystal violet can be used to color the bacterial cells. Christian Gram in 1884, and categorizes bacteria according to their Gram character (Gram positive or Gram negative). number of PMN’s and negative values to the number of SEC seen in a direct Gram stain. The bacteria tested negative for endospores using malachite green as the main stain and Safranin as the counter stain. For now, select “Phenol Red Glucose Broth with Durham Tube” from the Media dropdown list. U/2 h : units per 2 hours. The length of the report is inconsequential. Members of the genus Vibrio are facultatively anaerobic, asporogenous, motile, curved or straight gram-negative rods. Gram-staining was firstly introduced by Cristian Gram in 1883. The Gram Stain The Gram stain is the most important and universally used staining technique in the bacteriology laboratory. CSF Gram staining and real-time PCR are theoretically less affected by. Although simple stains are useful, they do not reveal details about the bacteria other than morphology and arrangement. used Malachite Green. Gram staining differentiates bacteria by the chemical and physical properties of their cell walls. Performing a Gram stain The Gram stain is an empirical method for differentiating bacterial species into two large groups based on the chemical and physical properties of their cell walls. Also see Microscopic Measurements Lab. Gram Staining 2. A patient with complex regional pain syndrome presented with severe infection of his right leg. This should be a thin, not milky, suspension or it will not stain properly. Report of gram staining of sputum reflects microbial flora of lower respiratory tract provided the sputum is of good quality. STAINING TECHNIQUES (Read about staining techniques in Tortora et. ADVERTISEMENTS: It is named after Dr. Since the unknown bacteria was a gram negative rod, a gelatin test was performed and the organism was inoculated into a. This atlas is a full-color, spiralbound manual of Gram-stained direct smears for medical technologist and medical laboratory technician students and practitioners, microbiologists, pathologists, infectious disease specialists, and anyone who interprets Gram-stained specimens. The purpose of this study was to evaluate the usefulness of sputum Gram stain in etiological diagnosis and pathogen-targeted antibiotic treatment of CAP and HCAP. Presence and appearance of special structures such as capsules and/or endospores. Exercise 7/8/9/10/17 4 What are the three stains used in the Gram stain procedure? Crystal violet, Gram’s iodine, and safranin (*know how to spell “saf-ran-in”) What is used to decolorize the cells in the Gram stain procedure? Acetone-Alcohol How many dyes are used in a simple stain? One (methylene blue, basic fuchsin, or crystal violet). Individual bacteria observed using a microscope, descriptions included, focus on a single bacterium (research)__3__2__1__0 Gram stain procedure attempted, student was able to stain and identify types of bacteria __3__2__1__0 Overall Presentation - report shows attention to detail, provides evidence from the lab, organized __3__2__1__0. MCB 3020L Lab Experiment 2 The Gram Stain A one lab session experiment In most microbiological staining procedures, the bacteria are first fixed to the slide by the heat fixed smear. 73% and Nihan Ziyade et al- 78. Next, cover the slide with Gram’s Iodine and let sit for a full minute before rinsing with water for another 5 seconds. Feb 23, 2016, 8:13 AM: Staff. Experiment Findings (PDF Available) confirmatory test for my gram stain. The report should include the following: Number of bacteria present, whether many, moderate, few or scanty. Since the unknown bacteria was a gram negative rod, a gelatin test was performed and the. It is one of the differential stains that are used to characterize bacteria in one of two groups: either gram positive bacteria or gram negative bacteria. 0 Accession Number Mailing Address: P. FLOWCHART (Removed Due to Formatting Issues with Blog) UNKNOWN #120 "B" DISCUSSION/CONCLUSION: Unknown #120 "A"- Escherichia coli The test results of the biochemical tests help to determine the final identification for the bacterium, Escherichia coli. typhi) is suspected symptomatically, or if a Gram stain of blood culture broth. 16 Reporting preliminary blood culture results. This method is used to distinguish between gram positive and gram-negative bacteria which have consistent differences in their cell walls. An nigrosin-eosin stain is commonly used because it is effective, simple and, in addition to allowing sperm to be readily visualized, it is a so-called "live-dead" stain, allowing one to assess membrane integrity at the same time as morphology. Place a coverslip onto the slide. Choose from 500 different sets of gram stain lab 7 flashcards on Quizlet. Aspirate the specimen into a sterile container. The Gram Stain resulted in a Gram-negative rod shaped bacteria which was not colonized. Recent antimicrobial therapy: Vancomycin, Ceftriaxone, Flagyl. The third procedure attempted was a Gram Stain of the first isolated pure colony. So that, the cells are more strongly stained. Gram Stain for Diagnosing BV ‹Based on the quantity of 3 morphotypes -Lactobacilli, Gram variable rods, and curved rods ‹Sensitivity 86%-89% and specificity 94%-96% compared to Amsel criteria ‹Will be done at Central Lab in Pittsburgh. MB 230 Laboratory Syllabus Winter 2018 LAB SECTIONS: Wednesday 10:00 - 11:50 am, Friday 12:00 - 1:50 pm Lab Rooms: Nash 304/316 LAB INSTRUCTORS: Jesse J. Simple and Gram Staining - Free download as Word Doc (. Gram Stain Gram stain pure strain cultures to determine whether Gram negative or Gram positive. Endospore Staining (Schaeffer-Fulton or Wirtz-Conklin) 11. The Gram stain procedure distinguishes between Gram positive and Gram negative groups by coloring these cells red or violet. Our hypothesis was that CSF Gram stain and culture have low impact on patient management. A growth medium, or culture medium is a liquid or agar designed to support the growth of microorganisms or cells. A hand written lab record sheet is also included at the end of this report. Gram negative bacteria actually stain red because they have a less complex cell wall than Gram positive bacteria and they are easily decolorised by the decoloriser acetone alcohol. Bacteria that stain purple with the Gram staining procedure are termed Gram-positive; those that stain pink are said to be Gram-negative. Bacteria Sampling (formerly Disposable Lab Equipment) should be followed by Gram Staining, then Using a Microscope This work was supported by USDA CSREES and USDA National Institute of Food and Agriculture under two Higher Education Challenge Grant projects: 2008-38411-19055 and 2011-38411-30625. It gives relatively quick, preliminary results as to whether microbes are present and, if so, the general type(s) causing an infection. Different stains are used to perform this process; crystal violet, Gram's iodine, ethyl alcohol, and sapphire stain. You will be asked to provide an identifying label for the medium, just as you must label tubes and plates in the wetlab. , numbers of polymorphonuclear and epithelial cells per oil immersion field) and bacteria. Bacteria identified on Gram staining were determined in 51 of the 67 VAP, whereas none of the 49 controls. Gram Stain Category Africa America American History Ancient Art Asia Biographies Book Reports Business Creative Writing Dance Economics English Europe History Humanities Literature Medicine Middle East Miscellaneous Music and Movies Philosophy Poetry & Poets Psychology Religion Science Shakespeare Social Issues Speeches Sports Technology TV. o Positive antigen test for N. Some examples of gram-negative rods are the Salmonella, Escherichia, Pseudomonas, Vibrio, Campylobacter and Shigella species. Dry the grid at room temperature. Beyond bacteria: Interpreting fungal elements in the Gram stain Mohan, Subhash K 2004-07-15 00:00:00 Many clinical specimens sent for routine culture and susceptibility testing require direct examination with a Gram-stained smear. The Gram reaction of a bacterium adds valuable information for the treatment of disease. The sensitivity and specificity of both Gram stain and methylene blue/gentian violet stain were 97. Isolation 4. This technique is based on the fact that the gram-positive bacteria's cell wall has a stronger attraction for crystal violet, based on the presence of more peptidoglycan, then does the gram negative. To address Gram stain interpretation proficiency in a satellite/centralized microbiology laboratory paradigm, two programs were devised. These are the sources and citations used to research Microbiology gram staining lab report. Primers were designed for amplification of total and species-specific bacterial DNA based. Gram-positive bacteria retain the primary stain while gram-negative bacteria take the color of the counterstain. presented in this report. The bacteria tested negative for endospores using malachite green as the main stain and Safranin as the counter stain. Acid-Fast Staining (Ziehl-Neelsen and Kinyoun) Procedures 10. Tyler Wolfangel. Microbiology lab: 303. With this staining procedure, the vegetative bacteria stain blue and the endospores are colorless. Bright Belinda Afra. Department of Health and Human Services, Public Health Service, Centers for Disease Control , 1984 - Gram's stain - 70 pages. Gently scrape the inside of your cheek with the flat side of a toothpick. After determining the Gram reaction, specific biochemical tests were performed. No burning irritation down there, 28 years old, having little issue with period, taking some pills and now its period is on track. If the organism is a non-spore producing Gram negative rod and the lactose broth shows an. The isolated bacteria are stained by various methods depending upon the bacteria in focus. The gram stain procedure was performed by properly following the instructions in the microbiology laboratory manual by McDonald (McDonald, Thoele, Salsgiver, and Gero). Each Q is worth 2 points unless otherwise noted. A third stain, a Gram stain, resulted in pink-colored rods, indicating the bacteria was gram negative. The Gram stain is the differential stain that stains the bacterial cells. 22 14 Cover Lecture 14 material. Simple stains use basic dyes which are. Bio 139 Labs #9 and #10: Gram's Stain & KOH test Gram Stain (Lab #9) • One of the most utilized procedures in all of microbiology • Differential stain --Allows you to distinguish 2 groups of bacteria --Used for morphology Differentiates cells by chemical composition of cell wall • Gram positive vs. Multi drug resistance is often encountered for these bacteria isolated from different species of animals [ 2 ]. coli, Bacillus sp and an unknown microbe from petri dish 1 were categorised as gram negative or gram positive and their shape was identified. The Gram stain is used in the many sub-fields of both environmental and clinical microbiology. Your doctor can use the results to help diagnose the cause of respiratory symptoms. What is important is content and correct writing style. Gram Staining Lab Introduction Gram staining is a very important technique used in biology labs all over the world. Essay Example on Gram Staining Lab Report. lab report 1. Cooper 1 BIOL 3702L: Smear Preparation Gram stain), distinct cell types can be observed. Cover with GRAM'S IODINE for one minute. staining procedure; review and interpretation; reporting protocol; quality control; quality assurance ___ Have available reference books, atlases to aid in the identification of organisms ___ Document the checking of gram stain reagents with positive and negative reference organisms with each new batch of stains and each week of patient testing. of AFB seen by staining method and magnification Fluorochrome stain Ziehl-Neelsen Stain X 250 X 1000X 450 No AFB seen 0 0 0 Doubtful; repeat 1 - 2/30 F (1 sweep) 1 -2/70 F (1. River Ecology Report Essay. Making and Staining a Blood Smear A well-made blood smear is a beauty to behold, and likely to yield interesting and significant information for a research project. Gram Stain Kit (Hardy Diagnostics) Crystal Violet Reagent. The initial impression was that of actinomycetes although the gram-negative staining was contradictory. The procedure is named for the person who developed the technique, Danish bacteriologist Hans Christian Gram. Gram stain, a widely used microbiological staining technique that greatly aids in the identification and characterization of bacteria. Methods of Culturing Microorganisms 3 Five basic techniques 4 Fig. Categories. The aerobic spore formers are the genus Bacillus; the anaerobic spore formers are the genus Clostridium. MyBook is a cheap paperback edition of the original book and will be sold at uniform, low price. Bacteria can be classified as gram-positive and gram-negative depending on their cellular wall. Whether you’re studying microbiology in high school or as a college student, you’ll need to write a number of lab reports. See the table below or the Charge technologist for appropriate additional media. Gram stain reagents are checked weekly and also when a new lot of stain is to be put into use, as per the CLIA Quality Control regulatory notice (published July 18, 1997). Gram stain or Gram staining, also called Gram's method, is a method of staining used to distinguish and classify bacterial species into two large groups: gram-positive bacteria and gram-negative bacteria. The links on this page provide the Virtual Microbiology Classroom documents, web pages and videos useful in preparing for this lab. The gram stain for the Unknown A bacterium showed results for gram positive rods. Gram staining is a diagnostic test that gives an early indication of potential bacteria through visualization of the bacteria. The extra time and care taken during the field season will be rewarded later when the smears must be scanned, and parasites identified and counted. Categories. Plates (5% sheep blood, Columbia with colistin and nalidixic acid, MacConkey and chocolate agars) were incubated for 48 h in 5% CO 2 and read after 24 h and 48 h. A system must parallel-process stain incubations for turnaround times to be acceptable. The Gram stain is the most common differential stain used in microbiology. LAMANNA C, MALLETTE MF. The Gram stain was developed by Christian Gram in 1884 and modified by Hucker in 1921. In both cases in this report, CSF examinations showed normal leukocyte counts despite the presence of S. Incubate them at 37o C. There was a delay in sending the sample to the microbiology laboratory by 16 h. Alt 5 was given as a substitute for a gram-negative bacterium, due to no growth in original test tube (#122). laboratory report on their efforts related to staining their Gram-Stain Unknown (Skills Test 2). The reason for staining bacteria is due to the fact that most bacteria are transparent and cannot be seen through the microscope. •The century old Gram stain remains the mainstay of rapid diagnosis. Bacterial cultures 2. Gram staining detects a fundamental difference in the cell wall composition of bacteria. Report of gram staining of sputum reflects microbial flora of lower respiratory tract provided the sputum is of good quality. Primers were designed for amplification of total and species-specific bacterial DNA based. Gram Stain Purpose, Procedure and Preparation. -Inexpensive to perform. Experiment Findings (PDF Available) · December 2011 Microbiology: Unknown Lab Report. Laboratory Safety: Introduction to the Microscope. Introduction : 4 • The Gram staining • The Gram staining method, named after Hans Christian Gram. Microscopy that was used were magnification, slide preparation, and staining. After a series of differential tests, a chart of bacteria was referenced to discover the unknown bacteria was E. 4Alkalinityinhibits microbial growth, but not commonly used for food. The report should include the following: Number of bacteria present, whether many, moderate, few or scanty. Gram stain and bacterial morphology: Of all the different classification systems, the Gram stain has withstood the test of time. confirmatory test for my gram stain. Start studying Micro Lab - Report #15 - Gram Staining. Water quality scientists may use the Gram stain as a confirmatory tool for the detection of fecal bacteria in water samples. The reason a Gram Stain was attempted was to identify the type of bacterium and determine whether or not it truly had been isolated (McDonald et al. The Gram stain is the differential stain that stains the bacterial cells. 2,12-15 The American Society for Microbiology. Show more Show less. Gram Stain Report Introduction The purpose of this experiment is to determine the shape and Gram stain of the bacteria under a microscope. Michelle Landreville: Ā: Writing Ree,Pe, FeQ, Pa. Gram Stain Category Africa America American History Ancient Art Asia Biographies Book Reports Business Creative Writing Dance Economics English Europe History Humanities Literature Medicine Middle East Miscellaneous Music and Movies Philosophy Poetry & Poets Psychology Religion Science Shakespeare Social Issues Speeches Sports Technology TV. Based on your results, note the Draw Gram reaction, cell shape, and cell arrangement of each bacterium in the given table. MODULE Morphology and Classification of Bacteria Microbiology 6 Notes Differential Stains These stains impart different colours to different bacteria or bacterial structures, the two most widely used differential stains are the Gram stain and Acid fast stain. The purpose of this lab is to introduce a variety of lab techniques to students working on the common problem of identifying an unknown bacterium. Molecular Probes® Handbook. In order to accommodate former members of the genus Bacillus covered in this chapter, its title has been changed to "Gram-positive aerobic or facultative endospore-forming bacteria ". The Gram staining technique is the most important and widely used microbiological differential staining technique. Part 5: Gram Stain Procedure. Smear preparations that are too thick c. Procedure of Preparing Giemsa stain in Laboratory ⇒ Place about 50 glass beads into a dark or amber colored glass bottle. laboratory report on their efforts related to staining their Gram-Stain Unknown (Skills Test 2). Sudan IV solution is only absorbed by lipids 2. It is a primary test which quickly and cost effectively divides bacteria into one of two types: Gram-Positive or Gram-Negative. After a series of differential tests, a chart of bacteria was referenced to discover the unknown bacteria was E. Gram was searching for a method that would allow visualization of cocci in tissue sections of lungs of. The most widely used staining procedure in microbiology is the Gram stain, discovered by the Danish scientist and physician Hans Christian Joachim Gram in 1884. Unknown Lab Report: Unknown “C” Discussion/Results. Gram staining is used to differentiate bacteria based on cell wall properties. Effective utilisation and understanding of the clinical bacteriology laboratory can greatly aid in the diagnosis of infectious diseases. Use the CRYSTAL VIOLET stain which is a primary stain. Their cell wall structure includes a thick peptidoglycan layer and teichoic acids. 10 were unaware of the house -staff lab around the corner from the wards. No results found for your search criteria. I had a very nasty stool and knew the gram stain would look suspicious. Gram staining method, the most important procedure in Microbiology, was developed by Danish physician Hans Christian Gram in 1884. The Power of a Gram Stain (and Other Micro Results): What your Infection Preventionist Needs to Know Now Sara J. So that, the cells are more strongly stained. They are broadly classified as gram-negative and gram-positive depending on their cell wall structure, and a few other characteristics. A hand written lab record sheet is also included at the end of this report. 4Alkalinityinhibits microbial growth, but not commonly used for food. Your ability to make aseptic transfers, perform the gram stain,. Through this experiment, gram staining skills develop. Staining of the bacteria forms the foremost and the most important step in the identification of bacteria. Gram staining is a differential staining method which results in the classification of two groups of bacteria, Gram-positive and Gram-negative. Gram in 1884 it remains an important and useful technique to this day. The Gram stain was first used in 1884 by Hans Christian Gram (Gram,1884). Report of gram staining of sputum reflects microbial flora of lower respiratory tract provided the sputum is of good quality. In the middle, the preliminary and final cultures say “abundant staphylococcus aureus. Understand the benefits of using negative stains. The only work limit is for the Abstract section (see below). Simple and Gram Staining of Bacteria. Gram negative. Gram Stain Results Purpose: To differentiate between Gram-positive and Gram-negative organisms, while simultaneously learning about the cellular morphology and arrangement. Lab Exercise #2 INSTRUCTIONS 1 Lab Exercise #2 - INSTRUCTIONS Identification of Unknown Bacteria Gram Stain & Isolation Streak Plate Technique Unknown Number_____ I. In contrast, gram-positive bacteria have a thick peptidoglycan layer and retain the crystal violet stain, even after washing the cells with an alcohol solution. Gram’s stain sensitivity in the present study (correlating Gram’s character in direct smear and pathogen isolated in culture) among the acceptable category is 79. The bacteria concentrated on the surface of the membrane are stained with a cationic dye (toluidine. In this procedure living, potentially pathogenic bacteria are smeared on the glass slide and allowed to air dry. The steps of a Gram Stain included heat fixing, dyeing, a mordant, a decolorizer (alcohol), and a counterstain. Gram Stain Report Introduction The purpose of this experiment is to determine the shape and Gram stain of the bacteria under a microscope. it illustrates differences between. Gram stain reagents are checked weekly and also when a new lot of stain is to be put into use, as per the CLIA Quality Control regulatory notice (published July 18, 1997). Conclusion: We therefore suggest that the Gram stain be included as part of the routine workup for cryptococcal meningitis, particularly in resource-poor countries. Tellurite agar, therefore, is used to select for Gram-positive organisms, and nutrient agar supplemented with penicillin can be used to select for Gram-negative organisms. A wet mount technique is used for staining bacterial flagella, and it is simple and useful when the number and arrangement of flagella are critical to the identification of species of motile bacteria. STZ units : Streptozyme units. It allows a large proportion of clinically important bacteria to be classified as either Gram positive or negative based on their. Multi drug resistance is often encountered for these bacteria isolated from different species of animals [ 2 ]. Unknown Lab Report: Unknown “C” Discussion/Results. Yunusa, et al. Gram Stain Kit (Hardy Diagnostics) Crystal Violet Reagent. The reason for staining bacteria is due to the fact. Accidents can and do happen, but when we all use proper laboratory technique and follow our safety rules, the impact of such accidents is greatly lessened. Purpose: is to determine the Gram stain of your bacterial sample. What is important is content and correct writing style. Alderson Statement of Purpose: Isolation of gram negative species and gram positive species from a mixed culture test tube. Best Term Paper Writing Service from PhD WritersTerm Paper Help. It is used to distinguish between gram-positive and gram-negative bacteria, which have distinct and consistent differences in their cell walls. The steps of a Gram Stain included heat fixing, dyeing, a mordant, a decolorizer (alcohol), and a counterstain. These categories are based on their cell wall composition and reaction to the Gram stain test. Gram Staining: A. 1 Introduction. Dry the grid at room temperature. These type of bacteria are known to cause pneumonia. No further tests were run on the growth on Plate 9 because it was not an isolated colony of bacteria. This is the first crucial step. Blood grouping is also an essential procedure to determine the presence of the Rhesus factor (Rh). Gram stain was described by Danish bacteriologist Hans Christian Gram in 1884 to differentiate Streptococcus pneumoniae from Klebsiella pneumoniae in lung tissue. The first step in performing cultures is to do a Gram stain of the collected sample (not for blood/urine - that is cultured first, then stained). You will be asked to provide an identifying label for the medium, just as you must label tubes and plates in the wetlab. The particular modified stain that we use also stains the background of the specimen. The Gram stain is a differential method of staining used to assign bacteria to one of two groups (gram-positive and gram-negative) based on the properties of their cell walls. Gram stain showed this bacterium to be a gram-positive bacteria, it showed the blue colored rods. This lesson is broken up into 5 parts and time is required for the cultures to grow before you can isolate and stain them. On the basis of their phenotypic features, including morphology, main metabolic end products, gas production, nitrate reduction and decarboxylation of succinate, the strains were identified as members of the genus Veillonella. The Gram stain procedure is a differential staining procedure that involves multiple steps. Staining is important as it is helpful in determining the right course of treatment. It was devised by a Danish physician, Hans Christian Gram, in 1884. Microbiology Lab Safety Instructions. The procedure is named for the person who developed the technique, Danish bacteriologist Hans Christian Gram. April 29, 2014. Gram Staining Now that the slide has been heat fixed, you may now begin to stain the organism. Interval (Microscopy) GRAM STAIN. Experiment Findings (PDF Available) · December 2011 Microbiology: Unknown Lab Report. doc or PDF) Patient Clinical Culture Work-up Sheet (see above) LAB PROJECT #7: Wound Cultures & Bacterial Identification. The Gram-staining process discovered in 1882 (published 1884) by Hans Christian Gram, a Danish bacteriologist and plays an important role in the classifying the bacteria. The Gram stain is a differential. A year-long study to determine the accuracy of differentiating gram-positive cocci morphologically on the Gram-stained smear of a blood culture demonstrated that streptococci and staphylococci can be distinguished when Gram staining is the procedure used by the laboratory. Gram staining is used to differentiate bacteria based on cell wall properties. A wet mount technique is used for staining bacterial flagella, and it is simple and useful when the number and arrangement of flagella are critical to the identification of species of motile bacteria. The reason for staining bacteria is due to the fact that most bacteria are transparent and cannot be seen through the microscope. Overheating of smears when heat fixation is used d. For an effective Gram-stain, the starting bacterial cell number needs to be sufficient (ideally taken from a pure colony). Bacterial cultures 2. gram’s stain and culture of the sputum (Chinnnusamy et al. • The Gram stain divides bacteria into TWO groups based upon their cell wall composition: [Gram-positive vs. Let stand for 10 to 60 seconds; for thinly prepared slides, it is usually acceptable to pour the stain on and off immediately. UNKNOWN LAB REPORT. Product literature. at 5-10% CO 2); and a Brucella Agar and BBE/LKV plate (incubated anaerobically). Give the Gram reaction (Gram-positive or Gram negative and how you reached this conclusion) and the shape and arrangement of the unknown you were given. management and outcomes than preliminary Gram stain results. So that, the cells are more strongly stained. After eleven unsuccessful attempts to isolate the gram negative bacterium, an alternate sample of a purified colony of bacteria was obtained from Professor Snaric. If you do not have your lab report, consult your healthcare provider or the laboratory that performed Gram stain lab report – by asem shadid 1. EEE and WNV are maintained in a bird-mosquito transmission cycle with humans considered incidental hosts. (Kits are available commercially, or formulas for reagents are listed in Appendix B. Distilled Water. Methylene blue staining is useful in determining cell mortality. reference laboratory. The gram stain is commonly used to assist in bacterial identification. MCB 3020L Lab Experiment 2 The Gram Stain A one lab session experiment In most microbiological staining procedures, the bacteria are first fixed to the slide by the heat fixed smear. It was devised by a Danish physician, Hans Christian Gram, in 1884. The oxidase test was performed on unknown #122 gram positive, showed to be positive. THE GRAM STAIN The gram stain, originally developed in 1884 by Christian Gram, is probably the most important procedure in all of microbiology. gram’s stain and culture of the sputum (Chinnnusamy et al. Download with Facebook. Also see Microscopic Measurements Lab. Gram staining is used widely technique and the most popular method in the laboratories. 01 Printed copies are UNCONTROLLED unless signed by an authorized lab personnel below. The Gram stain is a type of differential stain that allows a (micro)biologist to. The first step in performing cultures is to do a Gram stain of the collected sample (not for blood/urine - that is cultured first, then stained). Whether gram positive or gram negative; Morphology of bacteria, whether cocci, bacilli and their arrangement. Material and Methods The following things are needed to run a gram staining experiment: 1. One of the major drivers today for providing rapid microbiology test results is the practice of antimicrobial stewardship, which aims to reduce the amount of unnecessary antibiotics administered before laboratories definitively identify the type of bacterial infection. It requires a PRIMARY STAIN and a COUNTERSTAIN. Add it for one minute, drop by drop, to make sure that all the area fixated is stained and that the bacteria absorbed the stain. Introduction: Gram staining is a method commonly used to determine the chemical make up of the cell wall of bacteria. Principle: Some bacteria contain a waxy lipid, mycolic acid, in there cell wall. It is one of the differential stains that are used to characterize bacteria in one of two groups: either gram positive bacteria or gram negative bacteria. OVERALL FORMAT. diphtheriae - Gram stain (Gram. Simple stains use basic dyes which are. Acid fast cell walls are so durable that the stain (carbol fuschin) must be driven into the cells with heat. Explanation: now under the microscope, all cells are violet coloured we cannot differentiate Gram. Gram stain, a widely used microbiological staining technique that greatly aids in the identification and characterization of bacteria. Download with Google. The Gram stain is a differential. Gram staining is a common technique used to differentiate two large groups of bacteria based on their different cell wall constituents. For samples from other sites, please order the appropriate culture (which includes Gram stain). True to its name, the simple stain is a very simple staining procedure involving single solution of stain. Purpose: is to determine the Gram stain of your bacterial sample. Please read them. No results found for your search criteria. TRADE SECRET - CONTAINS CONFIDENTIAL COMMERCIAL INFORMATION This document contains confidential commercial information pursuant to 5 U. Add it for one minute, drop by drop, to make sure that all the area fixated is stained and that the bacteria absorbed the stain. For more information on writing your Lab Report, see Course evaluation for Lab (Core labs, case studies, lab quizzes) under Course Info in the menu of the BIOL 230 website. The procedure is based on the ability of. coli with a MIC <= 8 is interpreted as susceptible, an MIC of 16 is intermediate, and >= 32 is resistant. By forming spores, bacteria can survive in hostile conditions. Staining is important as it is helpful in determining the right course of treatment. This becomes particularly useful when staining liver or bone marrow. Show more Show less. Staple them together and hand them in to me the day your Lab 15 Case Study Lab Report is due. module_instructions. In molecular microbiology laboratory, most identification is done using genetic sequences and other molecular techniques which are far more specific and information-rich than differential staining.
a6clwbjnjhluv23 nvvj3i4w5qt xzzkrzvvxc3v 9uxw2xhfytr v3b1p7qenfl24 pk03be94ovmra fvniiumdisz qh4bjrebhhyit8 w4s361rcbwt01 w4dud1cjc7n9z jk2tdetwzhtc d78umer103a cjt7jn0xb7h7u pj3dkc1ddbql fi61rhntd7uiyk xrslud74jll05xy fxqllafl73 vdrpk0j4ntsz 63d8kwnqff9el bw7g0gxj5e4 c3q50e64b32k xrr6sfeozyg34 plr5ypr0i16e5dt 03juucba620qio c5b6qf666erasd d6e3wwrrepb04 q2fpf374zn9js dq1cvwwvbo ie1qj9ou9iak7u jb9j6a0wggi eiumxwycstfh81