scholarly journals Assessing Gram-stain error rates within the pharmaceutical microbiology laboratory

2020 ◽  
Author(s):  
Tim Sandle
Keyword(s):  
Error Rates ◽  
Microbiology Laboratory ◽  
Gram Stain ◽  
Clinical Context ◽  
Determinative Bacteriology ◽  
Gram Positive ◽  
Gram Negative ◽  
Microbial Identification ◽  
Gram Staining ◽  
Gram Negative Organisms

Gram-staining remains the fundamental method for determinative bacteriology, dividing bacteria into Gram-positive and Gram-negative organisms. This test provides information as to the origin of any contamination and is a pre-requisite for many microbial identification methods. Despite the longevity of the test, the test is highly reliant upon analyst technique and therefore errors occur. While there are a few studies looking at errors in the clinical context, research has not been extended to the pharmaceutical microbiology laboratory context. In this study, we present a review of over 6,000 Gram-stains and establish an error rate of around 3%, with the most common reason for error being an over-decolourisation step resulting in organisms that should be Gram-positive appearing as Gram-negative. The analysis enables others to benchmark their facilities against.

scholarly journals Misinterpretation of Gram Stain from the Stationary Growth Phase of Positive Blood Cultures for Brucella and Acinetobacter Species

2017 ◽  
Vol 11 (1) ◽  
pp. 126-131 ◽  
Author(s):  
Ali M. Bazzi ◽  
Jaffar A. Al-Tawfiq ◽  
Ali A. Rabaan
Keyword(s):  
Real Time Pcr ◽  
Blood Cultures ◽  
Blood Agar ◽  
Gram Stain ◽  
Gram Positive ◽  
Gram Negative ◽  
Gram Staining ◽  
Urease Test ◽  
Oxidase Test ◽  
Vitek 2

Introduction:Acinetobacter baumanniiandBrucellaspecies are Gram-negative organisms that are vulnerable to misinterpretation as Gram-positive or Gram-variable in blood cultures.Objective:We assess the random errors in gram stain interpretation to reduce the likelihood of such errors and therefore patient harm.Methodology:Aerobic and anaerobic blood cultures from two patients in an acute care facility in Saudi Arabia were subjected to preliminary Gram-staining. In case 1, VITEK-2 Anaerobe Identification, repeat Gram staining from a blood agar plate, Remel BactiDrop™ Oxidase test, Urea Agar urease test and real-time PCR were used to confirm presence ofBrucellaand absence ofCoryneformspecies. In case 2, repeat Gram- staining from the plate and the vials, VITEK-2 Gram-Negative Identification, real-time PCR and subculture on to Columbia agar, blood agar, and MacConkey agar were carried out to identifyA. baumannii.Results:In case 1, initially pleomorphic Gram-positive bacteria were identified.Coryneformspecies were suspected. Tiny growth was observed after 24 h on blood agar plates, and good growth by 48 h. Presence ofBrucellaspecies was ultimately confirmed. In case 2, preliminary Gram-stain results suggested giant Gram-positive oval cocci. Further testing over 18-24 h identifiedA. baumannii.Conclusions:Oxidase test from the plate and urease test from the culture vial is recommended after apparent identification of pleomorphic Gram-positive bacilli from blood culture, once tiny growth is observed, to distinguishBrucellafromCorynebacteriumspecies. If giant Gram-positive oval cocci are indicated by preliminary Gram-staining, it is recommended that the Gram stain be repeated from the plate after 4-6 h, or culture should be tested in Triple Sugar Iron (TSI) medium and the Gram stain repeated after 2-4 h incubation.

Gram staining v1

2021 ◽  
Author(s):  
lydiariver not provided
Keyword(s):  
Cell Walls ◽  
Group B Streptococcus ◽  
Gram Negative Bacteria ◽  
Gram Positive ◽  
Gram Negative ◽  
Gram Staining ◽  
Acetone Treatment ◽  
Gram Negative Organisms ◽  
Group B ◽  
Gram Positive Cocci

Gram staining is one of the first techniques used for the identification of group B Streptococcus agalactiae and one would expect to see gram-positive cocci under the microscope. The technique consists of applying a series of colorants and bleaches (acetone), which interact with the lipids of the membranes of gram-positive and gram-negative bacteria. The cell walls of gram-positive organisms retain the dye after acetone treatment and appear purple in color, whereas gram-negative organisms become discolored after acetone treatment and appear pink.

scholarly journals POLA KUMAN BERDASARKAN PEWARNAAN GRAM PADA TINJA ANAK DENGAN DIARE DI RSUP PROF. DR. R. D. KANDOU MANADO

e-CliniC ◽  
2014 ◽  
Vol 2 (1) ◽  
Author(s):  
I Made Dwi Pramana
Keyword(s):  
Bacterial Infection ◽  
Microscopic Examination ◽  
Gram Negative Bacteria ◽  
Gram Stain ◽  
Gram Positive ◽  
Cross Sectional ◽  
Gram Negative ◽  
Consecutive Sampling ◽  
Gram Staining ◽  
In Infants And Children

Abstract: Background: Bacterial infection is one of the causes of diarrhea in infants and children. Bacteria are a group of microorganisms belongs to the prokaryotes which are structurally simpler than eukaryotics. There were many examinations used to detect the bacteria caused diarrhea, one of them was microscopic examination of the Gram stain smear. This technique is to determine whether the examination is Gram positive or Gram negative bacteria. Objective: This study aimed to determine the pattern of bacteria by Gram stain in children’s feces with diarrhea. Methods: This research used a descriptive designed with cross sectional approach by consecutive sampling from November to December 2013. There were 50 children in this study. Result: The results showed, 29 girls (58%) and 21 boys (42%). There were 34 children (68%) as the largest group that belongs to 1 - <3 years old. The results of feces microscopic examination showed 29 children (58%) got contaminated by bacteria. 23 children (46%) were contaminated with Gram positive and Gram negative bacteria. Gram negative Basil bacteria ware the most common bacteria that found in 23 preparations. Conclusion: Gram-negative bacilli germ were the most common germs that found in children’s feces with diarrhea and the numbers of diarrhea on November to December 2013 were increased. Key words: bacterial pattern, Gram staining, children, diarrhea  Abstrak: Latar belakang: Infeksi bakteri merupakan salah satu penyebab terjadinya diare pada bayi dan anak. Bakteri merupakan mikroorganisme yang termasuk dalam golongan prokariot yang strukturnya lebih sederhana dari eukariot. Banyak pemeriksaan yang dilakukan untuk mendeteksi bakteri penyebab diare, salah satunya dengan pemeriksaan mikroskopis pulasan yaitu pewarnaan Gram yang merupakan salah satu teknik pemeriksaan untuk menentukan apakah termasuk bakteri Gram positif atau bakteri Gram negatif. Tujuan penelitian: Penelitian ini bertujuan unutk mengetahui pola kuman berdasarkan pewarnaan Gram pada tinja anak dengan diare. Metode: Penelitian ini menggunakan desain penelitian deskriptif dengan pendekatan cross sectional yang dilakukan dengan cara consecutive sampling dari bulan November sampai Desember 2013. Sampel penelitian berjumlah 50 anak dan dilakukan pemeriksaan pewarnaan Gram. Hasil penelitian: Hasil penelitian didapatkan, perempuan 29 anak (58%) dan Laki-laki 21 anak (42%). Kelompok umur terbanyak 1 ─ <3 berjumlah 34 anak (64%). Hasil pemeriksaan mikroskopis feses ditemukan positif bakteri sebanyak 29 anak (58%). Bakteri Gram positif dan Gram negatif didapatkan berjumlah 23 anak (46%). Bakteri Basil Gram negatif merupakan bakteri terbanyak yang ditemukan yaitu 23 preparat. Kesimpulan: Kuman basil Gram negatif merupakan kuman terbanyak yang ditemukan pada tinja anak dengan diare dan terjadi peningkatan angka kejadian diare pada bulan November – Desember 2013. Kata kunci : pola kuman, pewarnaan Gram , anak, diare

scholarly journals A Fluorescent Gram Stain for Flow Cytometry and Epifluorescence Microscopy

1998 ◽  
Vol 64 (7) ◽  
pp. 2681-2685 ◽  
Author(s):  
David J. Mason ◽  
Subo Shanmuganathan ◽  
Fiona C. Mortimer ◽  
Vanya A. Gant
Keyword(s):  
Flow Cytometry ◽  
Epifluorescence Microscopy ◽  
Nucleic Acid Binding ◽  
Gram Stain ◽  
Green Fluorescence ◽  
Gram Positive ◽  
Gram Negative ◽  
Syto 13 ◽  
Gram Negative Organisms ◽  
Green Fluorescent

ABSTRACT The fluorescent nucleic acid binding dyes hexidium iodide (HI) and SYTO 13 were used in combination as a Gram stain for unfixed organisms in suspension. HI penetrated gram-positive but not gram-negative organisms, whereas SYTO 13 penetrated both. When the dyes were used together, gram-negative organisms were rendered green fluorescent by SYTO 13; conversely, gram-positive organisms were rendered red-orange fluorescent by HI, which simultaneously quenched SYTO 13 green fluorescence. The technique correctly predicted the Gram status of 45 strains of clinically relevant organisms, including several known to be gram variable. In addition, representative strains of gram-positive anaerobic organisms, normally decolorized during the traditional Gram stain procedure, were classified correctly by this method.

BACTERIAL LIPIDS AND THE GRAM STAINING REACTION

1964 ◽  
Vol 42 (8) ◽  
pp. 1141-1151 ◽  
Author(s):  
A. K. Bergh ◽  
S. J. Webb ◽  
C. S. McArthur
Keyword(s):  
Staphylococcus Epidermidis ◽  
Staining Reaction ◽  
Bacterial Cells ◽  
Polar Solvents ◽  
Gram Positive ◽  
Gram Negative ◽  
Gram Staining ◽  
A Cell ◽  
Gram Negative Organisms ◽  
High Polarity

In this study, no correlation was found between the Gram staining reaction of bacterial cells and the kinds of fatty acids in the lipids. The polar solvent (usually alcohol) used to wash out the stain from Gram-negative organisms removes much more lipid from these than from Gram-positive forms. The extractability of lipids from cells of Staphylococcus epidermidis increased when they became Gram-negative through aging. The infrared spectrum of the lipids extracted from these Gram-negative Staphylococci remained the same as that of the Gram-positive cells. It was further noted that high polarity characteristics and mutual miscibility in aqueous and lipid solvents are requirements of the decolorizing solvent.The Gram staining reaction of a cell appears to be related to the manner in which some of its lipids are bound to other components of the cell and the ease or difficulty with which the lipids are separated from lipoprotein and other complexes by certain polar solvents.

scholarly journals Linearized esculentin-2EM shows pH dependent antibacterial activity with an alkaline optimum

2021 ◽  
Author(s):  
Erum Malik ◽  
David A. Phoenix ◽  
Timothy J. Snape ◽  
Frederick Harris ◽  
Jaipaul Singh ◽  
...  
Keyword(s):  
Helical Structure ◽  
Food Preservation ◽  
Forming Process ◽  
Gram Positive ◽  
Gram Negative ◽  
Bacterial Membranes ◽  
Gram Positive Bacteria ◽  
Alkaline Conditions ◽  
Ph Dependent ◽  
Gram Negative Organisms

AbstractHere the hypothesis that linearized esculentin 2EM (E2EM-lin) from Glandirana emeljanovi possesses pH dependent activity is investigated. The peptide showed weak activity against Gram-negative bacteria (MLCs ≥ 75.0 μM) but potent efficacy towards Gram-positive bacteria (MLCs ≤ 6.25 μM). E2EM-lin adopted an α-helical structure in the presence of bacterial membranes that increased as pH was increased from 6 to 8 (↑ 15.5–26.9%), whilst similar increases in pH enhanced the ability of the peptide to penetrate (↑ 2.3–5.1 mN m−1) and lyse (↑ 15.1–32.5%) these membranes. Theoretical analysis predicted that this membranolytic mechanism involved a tilted segment, that increased along the α-helical long axis of E2EM-lin (1–23) in the N → C direction, with −  < µH > increasing overall from circa − 0.8 to − 0.3. In combination, these data showed that E2EM-lin killed bacteria via novel mechanisms that were enhanced by alkaline conditions and involved the formation of tilted and membranolytic, α-helical structure. The preference of E2EM-lin for Gram-positive bacteria over Gram-negative organisms was primarily driven by the superior ability of phosphatidylglycerol to induce α-helical structure in the peptide as compared to phosphatidylethanolamine. These data were used to generate a novel pore-forming model for the membranolytic activity of E2EM-lin, which would appear to be the first, major reported instance of pH dependent AMPs with alkaline optima using tilted structure to drive a pore-forming process. It is proposed that E2EM-lin has the potential for development to serve purposes ranging from therapeutic usage, such as chronic wound disinfection, to food preservation by killing food spoilage organisms.

scholarly journals 1554. Epidemiology of Adult Bacterial Hand Infections at Two Urban Hospitals

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S777-S778
Author(s):  
Arsheena Yassin ◽  
Christine Stavropoulos ◽  
Krystina L Woods ◽  
Jiashan Xu ◽  
Justin Carale ◽  
...  
Keyword(s):  
New York ◽  
Staphylococcus Aureus ◽  
Antibiotic Regimen ◽  
Gram Positive ◽  
Gram Negative ◽  
Streptococcus Species ◽  
Urban Hospitals ◽  
Haemophilus Parainfluenzae ◽  
Empiric Antibiotic ◽  
Gram Negative Organisms

Abstract Background Hand infections represent a major source of morbidity, which can result in hand stiffness and amputation. Early appropriate empiric antibiotic regimen may reduce the associated morbidity, hence the importance to examine local epidemiology. The aim of this study was to define the current epidemiology of adult hand infections at two urban hospitals in New York City. Methods We performed a double center, retrospective study of adult patients hospitalized from March 2018 to May 2020. Patients with positive cultures associated with the hand infections were included. Retrospectively, 100 patients were reviewed. Data on baseline demographic, clinical, surgical, microbiology, and treatment parameters were collected. Results Of the 100 patients, 76% were male, with median age of 47.5 years (35, 58.25) and average C-reactive protein (CRP) of 50.66 mg/L (± 64.64) on admission (see Table 1). Previous hospitalization within 1 year (38%), previous surgical procedures (39%) and recent IV medication use (26%) were common. 130 bacterial isolates were identified (see Table 2). The most frequent organisms were Gram-positive, with Methicillin susceptible Staphylococcus aureus (MSSA, 25.38%), Streptococcus species (20.08%), and Methicillin resistant Staphylococcus aureus (MRSA, 15.38%) being the most common. Gram-negative organisms were infrequent, with Haemophilus parainfluenzae (3.85%), Enterobacter cloacae (3.85) and Pseudomonas aeruginosa (3.08%) being the most prevalent. Of the 100 patients, 27% had polymicrobial infections, associated with trauma (6%), illicit IV use (6%) and unknown (7%) etiologies. Table 1: Baseline demographics and co-morbid conditions Table 2: Types and numbers of organisms in relation to etiologies Conclusion Within our population, the most common organisms associated with hand infections were Gram-positive, with Staphylococcus aureus and Streptococcus species being the most prevalent. Gram-negative pathogens were infrequently isolated. The results within this study can provide guidance to clinicians on assessing the appropriate empiric antibiotic regimen in patients with hand infections, and can serve as a basis for further studies identifying risk factors associated with isolation of organisms associated with hand infections. Disclosures All Authors: No reported disclosures

scholarly journals Evaluation of MicroScan Bacterial Identification Panels for Low-Resource Settings

Diagnostics ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 349
Author(s):  
Sien Ombelet ◽  
Alessandra Natale ◽  
Jean-Baptiste Ronat ◽  
Olivier Vandenberg ◽  
Liselotte Hardy ◽  
...  
Keyword(s):  
Bacterial Species ◽  
Ease Of Use ◽  
Bacterial Identification ◽  
Gram Positive ◽  
Gram Negative ◽  
Low Resource Settings ◽  
Low Resource ◽  
Bacillus Spp ◽  
Gram Negative Organisms ◽  
Instructions For Use

Bacterial identification is challenging in low-resource settings (LRS). We evaluated the MicroScan identification panels (Beckman Coulter, Brea, CA, USA) as part of Médecins Sans Frontières’ Mini-lab Project. The MicroScan Dried Overnight Positive ID Type 3 (PID3) panels for Gram-positive organisms and Dried Overnight Negative ID Type 2 (NID2) panels for Gram-negative organisms were assessed with 367 clinical isolates from LRS. Robustness was studied by inoculating Gram-negative species on the Gram-positive panel and vice versa. The ease of use of the panels and readability of the instructions for use (IFU) were evaluated. Of species represented in the MicroScan database, 94.6% (185/195) of Gram-negative and 85.9% (110/128) of Gram-positive isolates were correctly identified up to species level. Of species not represented in the database (e.g., Streptococcus suis and Bacillus spp.), 53.1% out of 49 isolates were incorrectly identified as non-related bacterial species. Testing of Gram-positive isolates on Gram-negative panels and vice versa (n = 144) resulted in incorrect identifications for 38.2% of tested isolates. The readability level of the IFU was considered too high for LRS. Inoculation of the panels was favorably evaluated, whereas the visual reading of the panels was considered error-prone. In conclusion, the accuracy of the MicroScan identification panels was excellent for Gram-negative species and good for Gram-positive species. Improvements in stability, robustness, and ease of use have been identified to assure adaptation to LRS constraints.

Sign in / Sign up

Export Citation Format

Share Document