WordNet

(microscopy) a dye or other coloring material that is used in microscopy to make structures visible
color for microscopic study; "The laboratory worker dyed the specimen"
color with a liquid dye or tint; "Stain this table a beautiful walnut color"; "people knew how to stain glass a beautiful blue in the middle ages"
produce or leave stains; "Red wine stains the table cloth"
a soiled or discolored appearance; "the wine left a dark stain" (同)discoloration, discolouration
marked or dyed or discolored with foreign matter; "a badly stained tablecloth"; "tear-stained cheeks"
having a coating of stain or varnish (同)varnished
the act of spotting or staining something (同)spotting, maculation
(histology) the use of a dye to color specimens for microscopic study
Danish physician and bacteriologist who developed a method of staining bacteria to distinguish among them (1853-1938) (同)Hans C. J. Gram
a metric unit of weight equal to one thousandth of a kilogram (同)gramme, gm, g

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(…で)…‘を'『汚す』,‘に'しみをつける《+『名』+『with』+『名』》 / 〈ガラス・木材・布など〉‘に'『着色する』 / (…で)〈人格・名声など〉‘を'汚す,‘に'傷をつける《+『名』+『with』+『名』》 / 汚れる,しみがつく / 〈C〉〈U〉(…についた)『しみ』,汚れ《+『on』+『名』》 / 〈U〉〈C〉着色剤,染料 / 〈C〉(人格・名声などに対する)汚点,傷《+『on』(『upon』)+『名』》

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出典(authority):フリー百科事典『ウィキペディア（Wikipedia）』「2015/07/18 16:16:16」(JST)

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A Gram stain of mixed Staphylococcus aureus (Staphylococcus aureus ATCC 25923, Gram-positive cocci, in purple) and Escherichia coli (Escherichia coli ATCC 11775, gram-negative bacilli, in red), the most common Gram stain reference bacteria

Gram staining, also called Gram's method, is a method of differentiating bacterial species into two large groups (gram-positive and gram-negative). The name comes from the Danish bacteriologist Hans Christian Gram, who developed the technique.

Gram staining differentiates bacteria by the chemical and physical properties of their cell walls by detecting peptidoglycan, which is present in a thick layer in gram-positive bacteria.^[1] In a Gram stain test, gram-positive bacteria retain the crystal violet dye, while a counterstain (commonly safranin or fuchsine) added after the crystal violet gives all Gram-negative bacteria a red or pink coloring.

The Gram stain is almost always the first step in the identification of a bacterial organism. While Gram staining is a valuable diagnostic tool in both clinical and research settings, not all bacteria can be definitively classified by this technique. This gives rise to gram-variable and gram-indeterminate groups as well.

History

The method is named after its inventor, the Danish scientist Hans Christian Gram (1853–1938), who developed the technique while working with Carl Friedländer in the morgue of the city hospital in Berlin in 1884. Gram devised his technique not for the purpose of distinguishing one type of bacterium from another but to make bacteria more visible in stained sections of lung tissue.^[2] He published his method in 1884, and included in his short report the observation that the typhus bacillus did not retain the stain.^[3]

Uses

Gram staining is a bacteriological laboratory technique^[4] used to differentiate bacterial species into two large groups (gram-positive and gram-negative) based on the physical properties of their cell walls.^[5] Gram staining is not used to classify archaea, formerly archaeabacteria, since these microorganisms yield widely varying responses that do not follow their phylogenetic groups.^[6]

The Gram stain is not an infallible tool for diagnosis, identification, or phylogeny, and it is of extremely limited use in environmental microbiology. It still competes with molecular techniques even in the medical microbiology lab. Some organisms are gram-variable (meaning they may stain either negative or positive); some organisms are not susceptible to either stain used by the Gram technique. In a modern environmental or molecular microbiology lab, most identification is done using genetic sequences and other molecular techniques, which are far more specific and informative than differential staining.

Gram staining has proven as effective a diagnostic tool as PCR, particularly with regards to gonorrhoea diagnosis in Kuwait. The similarity of the results of both Gram stain and PCR for diagnosis of gonorrhea was 99.4% in Kuwait.^[7]

Medical

Gram stains are performed on body fluid or biopsy when infection is suspected. Gram stains yield results much more quickly than culturing, and is especially important when infection would make an important difference in the patient's treatment and prognosis; examples are cerebrospinal fluid for meningitis and synovial fluid for septic arthritis.^[4]^[8]

Staining mechanism

Gram-positive bacteria have a thick mesh-like cell wall made of peptidoglycan (50–90% of cell envelope), and as a result are stained purple by crystal violet, whereas gram-negative bacteria have a thinner layer (10% of cell envelope), so do not retain the purple stain and are counter-stained pink by the Safranin. There are four basic steps of the Gram stain:

Applying a primary stain (crystal violet) to a heat-fixed smear of a bacterial culture. Heat fixation kills some bacteria but is mostly used to affix the bacteria to the slide so that they don't rinse out during the staining procedure.
The addition of iodide, which binds to crystal violet and traps it in the cell,
Rapid decolorization with ethanol or acetone, and
Counterstaining with safranin.^[9] Carbol fuchsin is sometimes substituted for safranin since it more intensely stains anaerobic bacteria, but it is less commonly used as a counterstain.^[10]

Crystal violet (CV) dissociates in aqueous solutions into CV+
and chloride (Cl−
) ions. These ions penetrate through the cell wall and cell membrane of both Gram-positive and Gram-negative cells. The CV+
ion interacts with negatively charged components of bacterial cells and stains the cells purple.

Iodide (I−
or I−
3) interacts with CV+
and forms large complexes of crystal violet and iodine (CV–I) within the inner and outer layers of the cell. Iodine is often referred to as a mordant, but is a trapping agent that prevents the removal of the CV–I complex and, therefore, color the cell.^[11]

When a decolorizer such as alcohol or acetone is added, it interacts with the lipids of the cell membrane. A gram-negative cell loses its outer lipopolysaccharide membrane, and the inner peptidoglycan layer is left exposed. The CV–I complexes are washed from the gram-negative cell along with the outer membrane. In contrast, a gram-positive cell becomes dehydrated from an ethanol treatment. The large CV–I complexes become trapped within the gram-positive cell due to the multilayered nature of its peptidoglycan. The decolorization step is critical and must be timed correctly; the crystal violet stain is removed from both gram-positive and negative cells if the decolorizing agent is left on too long (a matter of seconds).

After decolorization, the gram-positive cell remains purple and the gram-negative cell loses its purple color. Counterstain, which is usually positively charged safranin or basic fuchsine, is applied last to give decolorized gram-negative bacteria a pink or red color.^[12]^[13]

Some bacteria, after staining with the Gram stain, yield a gram-variable pattern: a mix of pink and purple cells are seen. The genera Actinomyces, Arthobacter, Corynebacterium, Mycobacterium, and Propionibacterium have cell walls particularly sensitive to breakage during cell division, resulting in gram-negative staining of these gram-positive cells. In cultures of Bacillus, Butyrivibrio, and Clostridium, a decrease in peptidoglycan thickness during growth coincides with an increase in the number of cells that stain gram-negative.^[14] In addition, in all bacteria stained using the Gram stain, the age of the culture may influence the results of the stain.

Examples

Gram-positive bacteria

Gram-positive bacteria generally have a single membrane (monoderm) surrounded by a thick peptidoglycan. This rule is followed by two phyla: Firmicutes (except for the classes Mollicutes and Negativicutes) and the Actinobacteria.^[5]^[15] In contrast, members of the Chloroflexi (green non-sulfur bacteria) are monoderms but possess a thin or absent (class Dehalococcoidetes) peptidoglycan and can stain negative, positive or indeterminate.^[5]^[15] Members of the Deinococcus-Thermus group, stain positive but are diderms with a thick peptidoglycan.^[5]^[15]

Historically, the gram-positive forms made up the phylum Firmicutes, a name now used for the largest group. It includes many well-known genera such as Bacillus, Listeria, Staphylococcus, Streptococcus, Enterococcus, and Clostridium. It has also been expanded to include the Mollicutes, bacteria like Mycoplasma that lack cell walls and so cannot be stained by Gram, but are derived from such forms.

Normally, if Gram stain is done on acid-fast bacteria, they show up as if they are gram-positive, mostly because of their thick cell wall.

Gram-negative bacteria

Gram-negative bacteria generally possess a thin layer of peptidoglycan between two membranes (diderms). Most bacterial phyla are gram-negative, including the cyanobacteria, spirochaetes, and green sulfur bacteria, and most Proteobacteria and Escherichia coli. (exceptions being some members of the Rickettsiales and the insect-endosymbionts of the Enterobacteriales).^[5]^[15]

Gram-indeterminate bacteria

Gram-indeterminate bacteria do not respond predictably to Gram staining and, therefore, cannot be determined as either gram-positive or gram-negative. They tend to stain unevenly, appearing partially gram positive and partially gram negative, or unstained by either crystal violet or safranin. Staining older cultures (over 48 hours) can lead to false Gram-variable results, probably due to changes in the cell wall with aging. Gram-indeterminate bacteria are best stained using acid-fast staining techniques. Examples include many species of Mycobacterium, including M. tuberculosis and M. leprae.^[16]^[17]

References

^ Bergey, David H.; John G. Holt; Noel R. Krieg; Peter H.A. Sneath (1994). Bergey's Manual of Determinative Bacteriology (9th ed.). Lippincott Williams & Wilkins. ISBN 0-683-00603-7.
^ Austrian, R. (1960). "The Gram stain and the etiology of lobar pneumonia, an historical note". Bacteriol. Rev. 24 (3): 261–265. PMC 441053. PMID 13685217.
^ Gram, HC (1884). "Über die isolierte Färbung der Schizomyceten in Schnitt- und Trockenpräparaten". Fortschritte der Medizin (in German) 2: 185–189. .
English translation in: Brock, T.D. (1999). Milestones in Microbiology 1546–1940 (2 ed.). ASM Press. pp. 215–218. ISBN 1-55581-142-6. .
Translation is also at: Brock, T.D. "Pioneers in Medical Laboratory Science: Christian Gram 1884". Hoslink. Retrieved 2010-07-27.
^ ^a ^b Ryan KJ, Ray CG (editors) (2004). Sherris Medical Microbiology (4th ed.). McGraw Hill. pp. 232–3. ISBN 0-8385-8529-9.
^ ^a ^b ^c ^d ^e Madigan, MT; Martinko J; Parker J (2004). Brock Biology of Microorganisms (10th ed.). Lippincott Williams & Wilkins. ISBN 0-13-066271-2.
^ Beveridge TJ (2001). "Use of the Gram stain in microbiology". Biotech Histochem 76 (3): 111–8. doi:10.1080/714028139. PMID 11475313.
^ El-Garnal, A.H., Al-Otaibi, S.R., Alshamali, A., Abdulrazzaq, A., Najem, N., & Fouzan, A.A. Polymerase chain reaction is no better than Gram stain for diagnosis of gonococcal urethritis. Indian Journal of Dermatology, Venereology, and Leprology, (2009); 75, 101.
^ Søgaard M, Nørgaard M, Schønheyder H (2007). "First notification of positive blood cultures: high accuracy of the Gram stain report". J Clin Microbiol 45 (4): 1113–7. doi:10.1128/JCM.02523-06. PMC 1865800. PMID 17301283.
^ Microbiology; J.G. Black Prentice Hall, 1993
^ http://www.med-chem.com/procedures/GRAMSTAIN.pdf^{[dead link]}
^ Llewellyn, Brian D. (May 2005). "StainsFile – Stain theory – What a mordant is not". Retrieved 2009-09-10.
^ Beveridge TJ, Davies JA (November 1983). "Cellular responses of Bacillus subtilis and Escherichia coli to the Gram stain". Journal of bacteriology 156 (2): 846–58. PMC 217903. PMID 6195148.
^ Davies JA, Anderson GK, Beveridge TJ, Clark HC (November 1983). "Chemical mechanism of the Gram stain and synthesis of a new electron-opaque marker for electron microscopy, which replaces the iodine mordant of the stain". Journal of bacteriology 156 (2): 837–45. PMC 217902. PMID 6195147.
^ Beveridge TJ (March 1990). "Mechanism of gram variability in select bacteria". Journal of bacteriology 172 (3): 1609–20. PMC 208639. PMID 1689718.
^ ^a ^b ^c ^d Don J. Brenner, Noel R. Krieg, James T. Staley (July 26, 2005) [1984]. George M. Garrity, ed. Introductory Essays. Bergey's Manual of Systematic Bacteriology 2A (2nd ed.). New York: Springer. p. 304. ISBN 978-0-387-24143-2. British Library no. GBA561951. ^{[page needed]}
^ Black, Jacquelyn (2012). Microbiology: Principles and exploration (8th ed.). John Wiley & Sons. p. 68. ISBN 978-0-470-54109-8.
^ Reynolds J1, Moyes RB, Breakwell DP (2009). "Differential staining of bacteria: acid fast stain". Current Protocols in Microbiology. Appendix 3: Appendix 3H. doi:10.1002/9780471729259.mca03hs15. PMID 19885935.

External links

Gram staining technique video

UpToDate Contents

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1. 細菌検査室におけるグラム染色および培養結果に対するアプローチ approach to gram stain and culture results in the microbiology laboratory
2. 細菌性肺炎の評価のための喀痰培養 sputum cultures for the evaluation of bacterial pneumonia
3. 臨床微生物学レビュー：髄膜感染症 clinical microbiology review meningeal infections
4. 臨床微生物学レビュー：気道感染症 clinical microbiology review respiratory tract infections
5. 月齢1ヶ月以上の小児における急性細菌性髄膜炎：臨床的特徴および診断 bacterial meningitis in children older than one month clinical features and diagnosis

English Journal

Studies on visual detection and surface modification testing of glass microfiber filter paper based biosensor.

Adiguzel Y1, Kulah H2.Author information 1METU-MEMS Research and Application Center, Middle East Technical University (METU), Ankara, Turkey. Electronic address: yadiguzel@mems.metu.edu.tr.2METU-MEMS Research and Application Center, Middle East Technical University (METU), Ankara, Turkey; Electrical and Electronics Engineering Department, Middle East Technical University (METU), Universiteler Mah., Dumlupinar Bulv. No: 1, 06800 Cankaya, Ankara, Turkey. Electronic address: kulah@metu.edu.tr.AbstractGlass microfibers are commonly used as biomolecule adsorption media, as structural or disposable components of the optical biosensors. While any improvement in these components are appreciated, utilizing basic tools of traditional approaches may lead to original sensor opportunities as simple, functional designs that can be easily disseminated. Following this pursuit, surface modification of glass microfiber paper surface was performed by 3-aminopropyltriethoxysilane (APTES) and resulting improvement in the cell entrapment capacity could be observed visually, only after Gram staining. Gram staining offered rapid validation of enhanced binding on the glass surface. The same APTES-modified samples were also tested for binding of complementary DNA sequences and the results were less straightforward due to the necessity of DNA visualization by using a fluorescent stain, YOYO-1. Accordingly, when there were no surface modification, DNA and YOYO-1 adsorbed readily on the glass microfiber filter paper, and prolonged the interaction between DNA and YOYO-1. YOYO-1 adsorption on glass could be recognized from the color profile of YOYO-1 emission. This phenomenon can be used to examine suitability of APTES coverage on glass surfaces since YOYO-1 emission can be distinguished by its glass adsorbed versus DNA-bound forms. Aptness of surface coverage is vital to biosensor studies in the sense that it is preceding the forthcoming surface modifications and its precision is imperative for attaining the anticipated interaction kinetics of the surface-immobilized species. The proposed testing scheme offered in this study secures the work, which is aimed to be carried out utilizing such sensing systems and device components.
Biosensors & bioelectronics.Biosens Bioelectron.2014 Apr 15;54:27-34. doi: 10.1016/j.bios.2013.10.042. Epub 2013 Oct 31.
Glass microfibers are commonly used as biomolecule adsorption media, as structural or disposable components of the optical biosensors. While any improvement in these components are appreciated, utilizing basic tools of traditional approaches may lead to original sensor opportunities as simple, funct
PMID 24240165

Pseudomonas sihuiensis sp. nov., isolated from a forest soil in South China.

Wu M1, Wen J, Chang M, Yang G, Zhou S.Author information 1Guangdong Institute of Eco-Environmental and Soil Sciences, Guangzhou, 510650, People's Republic of China.AbstractA Gram-stain negative, motile, rod-shaped bacterium, designated strain WM-2(T), was isolated from a forest soil in Sihui City, South China, and characterized by means of a polyphasic approach. Growth occurred with 0-5 % (w/v) NaCl (optimum 0-1 %) and at pH 5.0-10.5 (optimum pH 8.5) and 4-40 °C (optimum 30 °C) in Luria-Bertani medium. Comparative 16S rRNA gene sequence analyses showed that strain WM-2(T) is a member of the genus Pseudomonas and most closely related to P. guguanensis, P. oleovorans subsp. lubricantis, P. toyotomiensis, P. alcaliphila and P. mendocina with 97.1-96.6 % sequence similarities. In terms of gyrB and rpoB gene sequences, strain WM-2(T) showed the highest similarity with the type strains of the species P. toyotomiensis and P. alcaliphila. The DNA-DNA relatedness values of strain WM-2(T) with P. guguanensis and P. oleovorans subsp. lubricantis was 48.7 and 37.2 %, respectively. Chemotaxonomic characteristics (the main ubiquinone Q-9, major fatty acids C18:1 ω7c/C18:1 ω6c, C16:0 and C16:1 ω7c/C16:1 ω6c and DNA G+C content 65.2 ± 0.7 mol%) were similar to those of members of the genus Pseudomonas. Polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, an unknown aminophospholipid, an unknown phospholipid and five unknown lipids. According to the results of polyphasic analyses, strain WM-2(T) represents a novel species in the genus Pseudomonas, for which the name Pseudomonas sihuiensis sp. nov. is proposed. The type strain is WM-2(T) (=KCTC 32246(T)=CGMCC 1.12407(T)).
Antonie van Leeuwenhoek.Antonie Van Leeuwenhoek.2014 Apr;105(4):781-90. doi: 10.1007/s10482-014-0134-3. Epub 2014 Feb 25.
A Gram-stain negative, motile, rod-shaped bacterium, designated strain WM-2(T), was isolated from a forest soil in Sihui City, South China, and characterized by means of a polyphasic approach. Growth occurred with 0-5 % (w/v) NaCl (optimum 0-1 %) and at pH 5.0-10.5 (optimum pH 8.5) and 4-40 °C (
PMID 24567079

Gramella planctonica sp. nov., a zeaxanthin-producing bacterium isolated from surface seawater, and emended descriptions of Gramella aestuarii and Gramella echinicola.

Shahina M1, Hameed A, Lin SY, Lee RJ, Lee MR, Young CC.Author information 1Department of Soil & Environmental Sciences, College of Agriculture and Natural Resources, National Chung Hsing University, Taichung, 402, Taiwan.AbstractA Gram-stain negative, strictly aerobic, zeaxanthin-producing, rod-shaped, non-spore-forming bacterial strain which is motile by gliding, designated CC-AMWZ-3(T), was isolated from surface seawater off coastal Kending, Taiwan. Strain CC-AMWZ-3(T) was found to share 93.3 % and 96.0-92.4 % pairwise 16S rRNA gene sequence similarity to Gramella echinicola KMM 6050(T) and other Gramella species, respectively, and formed distinct phyletic lineage during phylogenetic analysis. The major fatty acids were identified as C16:0, iso-C15:0, anteiso-C15:0, C16:1 ω6c and/or C16:1 ω7c and iso-C17:1 ω9c and/or C16:0 10-methyl. Polar lipids were found to include phosphatidylethanolamine, six unidentified lipids and three unidentified aminolipids. The DNA G+C content was determined to be 40.6 mol%. Menaquinone-6 was the sole respiratory quinone identified and triamine-sym-homospermidine was the predominant polyamine. Based on the polyphasic characteristics that are in line with those of Gramella species, in addition to distinguishing phylogenetic and phenotypic features, strain CC-AMWZ-3(T) appears to represent a novel species of the genus Gramella, for which the name Gramella planctonica sp. nov. (type strain CC-AMWZ-3(T) = JCM 18807(T) = BCRC 80553(T)) is proposed. In addition, emended descriptions of the species Gramella aestuarii and Gramella echinicola are also proposed.
Antonie van Leeuwenhoek.Antonie Van Leeuwenhoek.2014 Apr;105(4):771-9. doi: 10.1007/s10482-014-0133-4. Epub 2014 Feb 13.
A Gram-stain negative, strictly aerobic, zeaxanthin-producing, rod-shaped, non-spore-forming bacterial strain which is motile by gliding, designated CC-AMWZ-3(T), was isolated from surface seawater off coastal Kending, Taiwan. Strain CC-AMWZ-3(T) was found to share 93.3 % and 96.0-92.4 % pairwise
PMID 24522286

Japanese Journal

学校体育で共用されるスポーツ設備及び用具に付着する菌の状態

上岡尚代,橋本和幸,伊藤マモル
了徳寺大学研究紀要 (9), 49-56, 2015
NAID 40020415371

悪性リンパ腫に対する化学療法中に遅延発育型螺旋菌を検出した1例

佐野尚子,村田哲也,別所裕二 [他],齋藤麻菜美,伊藤竜吾,濱田正行
日本農村医学会雑誌 63(5), 764-771, 2015
　遅延発育型の菌は, 数日の血液培養で陽性シグナルを認めても培養液のグラム染色で見落とされやすく, 偽陽性として報告されてしまうことがある。今回我々は悪性リンパ腫に対する化学療法中の患者から遅延発育型螺旋菌が検出された症例を経験したので報告する。本症例では, 度重なる発熱に対して血液培養が施行され, 血液培養装置BACTEC9240 (Becton Dickinson, 以下BD) でボトル内CO …
NAID 130005063339

症例報告原発性肺クリプトコッカス症の1例 : 肺組織塗抹グラム染色による菌体像と形態計測結果について

沖本彩香,園部宏,三浦香苗 [他]
広島臨床検査 = Hiroshima journal of medical laboratory technology 3, 15-18, 2014
NAID 40020386913

Related Pictures

★リンクテーブル★

リンク元	「グラム染色」「Gram's staining」
拡張検索	「Gram staining」
関連記事	「stain」

「グラム染色」

　　[★]

英: Gram stain
同: (国試)Gram染色
関: 染色法

構成高分子	グラム陽性菌	グラム陰性菌
外膜	なし	あり
ペリプラズム	なし	あり
蛋白質の分泌場所	細胞外	ペリプラズムないし細胞外
ペプチドグリカン	多重層	単層ないし薄層
タイコ酸	あり	なし
リポ多糖	なし	あり(外膜)
リポタンパク質	まれにしかない	多くの場合存在する
リン脂質	なし	あり