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Bacterial Counts in Spleen
脾脏中的细菌菌落计数   

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Abstract

Bacterial loads can be determined as colony forming units (CFU) at any point of the infection by culturing spleen homogenates on agar plates. This is a reliable method for comparing the kinetics of infection in various mouse strains, estimating the virulence of different bacterial mutants or isolates and for vaccine testing and vacine estandarization. Although this method has been designed to recover Brucella or Salmonella organisms from spleen, the procedure may be applicable for other bacteria such as Listeria and Mycobacterium as well as to count bacterial loads in other organs such as liver or lymph nodes.

Keywords: Spleen(脾), CFU(CFU), Bacteria(细菌), Mouse(鼠标)

Materials and Reagents

  1. Sterile plastic bags, 30 g capacity (~30 ml) (Whirl-Pak® Write-On Bags) (Nasco, catalog number: B01067WA )
  2. 70% Ethanol (Merck KgaA, Emsure®, catalog number: 1009834000 )
  3. Latex examination gloves (Dermagrip®, catalog number: D1402-14 )
  4. Pyrogen-free Type 1+ grade destilled water (Milli-Q Direct 8 and 16 Ultrapure Water System)
  5. Dulbecco′s Phosphate Buffered Saline (Gibco, catalog number: 21300-058 )
  6. Tween® 20 (Sigma-Aldrich, catalog number: P5927 )
  7. Tryptone Soya Agar (Oxoid Limited, catalog number: CM0131 ) (see Recipes), or other media according to culture bacterial requirements
  8. PBS 0.1% Tween 20 (see Recipes)

Equipment

  1. 24-well plates, low binding cell (Sigma-Aldrich, catalog number: Z721077 )
  2. Beaker (100 ml)
  3. Glass alcohol burner (WHEATON, catalog number: WHE-237070 )
  4. Surgical instruments for mice (Mouse Surgical Kit) (Kent Scientific, model: INSMOUSEKIT )
  5. Drigalski spatula (Thermo Fisher Scientific, catalog number: NC0242405 )
  6. Micropipettes (10-100 μl and 100-1,000 μl)
  7. Micropipette tips (100 μl and 1 ml)
  8. 1.5 ml eppendorf tubes (Eppendorf, catalog number: 0 22363204 )
  9. Tuberculin plastic syringe (1 ml) with 25-27 g needle gauge
  10. Plastic petri dishes (90 x 15 mm)
  11. Disposable medical plastic gloves (Lab Depot, catalog number: 266-P )
  12. Small sharp scissors  and forceps
  13. Water purification system (Milli-Q Direct 8 and 16 Ultrapure Water System) (EMD Millipore, model: ZR0Q00800 )
  14. Bacterial incubator (Forma® Direct Heat CO2 Incubator) (Thermo Fisher Scientific, model: 310 )
  15. Autoclave (Yamato Scientific, model: SQ-500C )
  16. Analytical balance electronic digital (± 0.0001 g)
  17. Laminar flow cabinet (Esco Global, Labculture® Class II, Type A2, model: LA2-3A2 )

Procedure

  1. Spleen extraction
    1. Latex examination gloves should be used throughout the entire procedure.
    2. Mice (Note 1) are infected by the corresponding via (e.g. intraperitoneally, intravenously, subcutaneously) using the recommended bacterial dose (e.g. 102, 103, 104) depending on the assay and virulence of the bacteria employed. Intraperitoneal infection is carried out with tuberculin 1 cc syringe, 25-27 g needle and maximum volume of 20 ml/kg.
      http://www.procedureswithcare.org.uk/intraperitoneal-injection-in-the-mouse
    3. Weight sterile plastic Whirl-Pak Bags (one per each mouse spleen) to the mg level.
    4. Spleen must be obtained immediately after sacrifice (Note 2). Following the corresponding infection period (e.g. 3-15 days), mice are killed by cervical elongation keeping the appropriate ethical protocols and regulations (e.g. cervical dislocation, CO2) (Nagy et al., 2006) (Note 3).
    5. After killing, spray the mouse exhaustively with 70% alcohol, and locate on a clean or sterile surface, within a safety vertical laminar flow hood.
    6. With small sharp scissors make a small cut into the skin below the belly bottom. Then open the entire abdominal cavity and cut the peritoneum and expose the spleen (situated in the left superior abdominal quadrant of the mouse) or the required organ, following recommended necropsy protocols (Covelli, 2013 http://eulep.pdn.cam.ac.uk/Necropsy_of_the_Mouse/printable.php) (Note 4) (Figure 1).


      Figure 1. Spleen extraction indicating: A. position of the mouse for necropsy, B. opening of the mouse skin, C. opening of the abdominal wall and D. spleen extraction (pointed with white arrow). Photographs taken from Covelli, 2013. For details, this work may be consulted at: http://eulep.pdn.cam.ac.uk/Necropsy_of_the_Mouse/printable.php (date consulted: 09/30/2013).

    7. Use small forceps to hold the spleen and then cut the hilum together with the gastrosplenic ligament to remove the spleen and locate inside a pre-weighted sterile plastic Whirl-Pak Bag (Note 5) (Figure 1).
    8. Weight the spleens inside the bags using a 4 digit scale balance. To obtain the spleen weight, subtract the value of the empty preweighted plastic bag.

  2. Spleen homogenization
    1. Add 9 parts of PBS containing 0.1% Tween 20 per g of spleen (dilution 1:10), assuming that the volume of 1 g of spleen corresponds to 1 ml of PBS (e.g. 0.5 grams of spleen and 4.5 ml of PBS 0.1% Tween 20).
      Note: Homogenization is easier if a small volume is used at the beginning of the homogenization procedure and then, the remaining volume is completed to reach 1:10 dilution.
    2. Spleen homogenization is proficiently carried out by squeezing the organ inside bag by hand.
      Note: In order to release the intracellular bacteria, spleen cells are disrupted by squeezing the spleen tissue in PBS containing 0.1% Tween 20.
    3. After homogenization of the samples, and depending on expected bacterial spleen colonization, decimal (or the required) dilutions are performed with 1x PBS (on 1.5 ml eppendorf tubes or 24 well plates).
      Note: Open the plastic bag carefully and widely (using steel wires) to avoid contamination of the pipette. For infections using 1 x 106 CFU of bacteria and ranging from 1-30 days, plate 102-105 dilutions.

  3. Plating
    1. Two plating methods are suggested:
      Method A
      Dispense three separate 20 μl drops of each dilution on Tryptone Soya Agar (or the recommended media for growing the tested bacterium) plates using two plates per sample (Figure 2A). Then, let the drops to be absorbed in the agar surface (do not spread out the drops) prior incubation (5-10 minutes).
      Method B
      Dispense one 100 μl in each agar plate and distribute the sample with a sterile Drigalski spatula until the inoculum is fully dispersed on the agar surface using two plates per sample (Figure 2B).  
      Notes:
      1. Method A is quicker and fewer agar plates are required since four dilutions can be positioned on each plate. In addition, up to three drops can be put for each dilution, lessening counting errors. A minimum of 500 bacteria per g of spleen can be detected if 101dilution is plated.
      2. Method B is slower and increases plate consuming but is more sensitive when low bacterial loads are expected. Colony counting is easier since an entire plate is used per dilution. A minimum of 100 bacteria per g of spleen can be detected if 101dilution is plated.
    2. Plates are incubated under the required conditions for the bacterial strain (e.g. 37 °C under 5% CO2 atmosphere for 2-3 days) until colony forming units (CFUs) are evident.
    3. Count CFU in the various dilutions. Only use the plates (and the corresponding dilutions) in which separate colonies are obtained (10-30 CFUs in Figure 2A or 30-300 CFUs in Figure 2B).
    4. Calculate spleen bacterial loads by multiplying CFUs to the corresponding dilution and by 50 in method A or by 10 in method B, respectively.  
    5. CFUs means can be calculated either by CFUs/spleen (total CFUs recovered) or alternatively by CFUs/gram of spleen (total CFUs recovered divided by total grams of spleen). While the first method does not take into consideration spleen inflammation (size), the second method corrects for spleen swelling. In either case, it is always recommended to plot the spleen weight in a separate graph.


      Figure 2. Sample plating methods  

Notes

  1. Preferably, female mice (male mice from different litters have the tendency to fight) with weights ranging between 18-22 grams. Weight/age ratio varies depending upon the strain of mice. Older mice have the tendency of display more weight variation. Differences in weights or ages should be avoided. It is recommended to work with groups of four mice in tandem. Mice should be in good health conditions before the experiment. In the case of using immunosuppressed mice, it is recommended to include a group of non-infected negative control mice to check for previously acquired bacterial or yeast infections.
  2. Bacterial counts in organs after natural death or after a protracted period of time are not recommended, due to microorganism contamination during the process of tissue decay.
  3. Other methods used for sacrifice are CO2 affixation or overdose of anesthesia with components such as isoflurane (inhaled > 15%) or propofol (intravenously > 50 mg/kg).
  4. Avoid the skin hair of the mouse to become in contact with the peritoneal cavity.
  5. The spleen is easily detached without abundant hemorrhage. Surgical instruments must be sterilized with alcohol and flamed prior spleen manipulation to avoid contamination. External fat should be removed from the spleen to avoid extra weight errors.

Recipes

  1. Tryptone Soya Agar
    Suspend 40 grams of medium in 1 L Pyrogen-free Type 1+ grade destilled water
    Heat with frequent agitation and boil for 1 minute to completely dissolve the medium
    Autoclave at 121 °C for 15 min
  2. PBS 0.1% Tween 20
    Dissolve the following in 800 ml Pyrogen-free Type 1+ grade destilled water H2O
    8 g of NaCl
    0.2 g of KCl
    1.44 g of Na2HPO4
    0.24 g of KH2PO4
    1 ml Tween 20
    Adjust pH to 7.4
    Adjust volume to 1 L with additional Pyrogen-free Type 1+ grade destilled water H2O
    Sterilize by autoclaving

Acknowledgments

We thank María-Jesús Grilló for helping in the standardization of this technique. This protocol was adapted from the following original published papers Barquero-Calvo et al. (2007) and Nagy et al. (2007), and from Covelli (2013). This work was funded by grants from FIDA-2006, FS-CONARE (UNA/UCR), MICIT/CONICIT (FI-487-09), 8-N-2005 and B/3456-1 (NeTropica), B/3456-2 (IFS), AGL2004-01162/GAN (Spain), CNRS and INSERM (France) and MASTERSWITCH projects (European Communities).

References

  1. Barquero-Calvo, E., Chaves-Olarte, E., Weiss, D. S., Guzman-Verri, C., Chacon-Diaz, C., Rucavado, A., Moriyon, I. and Moreno, E. (2007). Brucella abortus uses a stealthy strategy to avoid activation of the innate immune system during the onset of infection. PLoS One 2(7): e631.
  2. Covelli, V. (2013). Guide to the Necropsy of the Mouse. 
    http://eulep.pdn.cam.ac.uk/Necropsy_of_the_Mouse/printable.php
  3. Nagy, A., Gertsenstein, M., Vintersten, K. and Behringer, R. (2006). Quick and humane sacrifice of a mouse by cervical dislocation. CSH Protoc 2006(1).    

简介

通过在琼脂平板上培养脾匀浆,可以在感染的任何点测定细菌负荷作为菌落形成单位(CFU)。 这是一种可靠的方法,用于比较不同小鼠品系中的感染动力学,估计不同细菌突变体或分离株的毒力,以及用于疫苗测试和疫苗预处理。 虽然该方法已经被设计为从脾脏中回收Brucella 或<沙门氏菌生物体,但是该方法可适用于其他细菌,例如李斯特氏菌 >分枝杆菌,以及计数其他器官如肝脏或淋巴结中的细菌量。

关键字:脾, CFU, 细菌, 鼠标

材料和试剂

  1. 无菌塑料袋,30g容量(〜30ml)(Whirl-Pak Write-On Bags)(Nasco,目录号:B01067WA)
  2. 70%乙醇(Merck KgaA,Emsure ,目录号:1009834000)
  3. 乳胶检查手套(Dermagrip ®,目录号:D1402-14)
  4. 无热原型1级脱脂水(Milli-Q Direct 8和16超纯水系统)
  5. Dulbecco's磷酸盐缓冲盐水(Gibco,目录号:21300-058)
  6. Tween 20(Sigma-Aldrich,目录号:P5927)
  7. 胰蛋白胨大豆琼脂(Oxoid Limited,目录号:CM0131)(参见Recipes)或根据培养菌需求的其它培养基。
  8. PBS 0.1%Tween 20(参见配方)

设备

  1. 24孔板,低结合细胞(Sigma-Aldrich,目录号:Z721077)
  2. 烧杯(100 ml)
  3. 玻璃酒精燃烧器(WHEATON,目录号:WHE-237070)
  4. 用于小鼠的外科器械(小鼠外科手术试剂盒)(Kent Scientific,型号:INSMOUSEKIT)
  5. Drigalski刮刀(Thermo Fisher Scientific,目录号:NC0242405)
  6. 微量移液器(10-100μl和100-1000μl)
  7. 微量吸头(100μl和1 ml)
  8. 1.5ml eppendorf管(Eppendorf,目录号:022363204)
  9. 结核菌素塑料注射器(1ml),具有25-27g针头规格
  10. 塑料培养皿(90×15mm)
  11. 一次性医用塑料手套(Lab Depot,目录号:266-P)
  12. 小锋利剪刀 和镊子
  13. 水净化系统(Milli-Q Direct 8和16超纯水系统)(EMD Millipore,型号:ZR0Q00800)
  14. 细菌孵育器(Thermo Fisher Scientific,型号:310)
  15. 高压灭菌器(Yamato Scientific,型号:SQ-500C)
  16. 分析天平电子数字(±0.0001 g)
  17. 层流柜(Esco Global,Labculture Class II,Type A2,型号:LA2-3A2)

程序

  1. 脾提取
    1. 在整个手术过程中应使用乳胶检查手套。
    2. 使用推荐的细菌剂量(例如 10 2 )通过相应的通道(例如腹膜内,静脉内,皮下)感染小鼠(注1) 10 3,<10> ),这取决于所使用的细菌的测定和毒力。用结核菌素1cc注射器,25-27g针和最大体积20ml/kg进行腹膜内感染。
      http://www.procedureswithcare.org.uk/intraperitoneal-injection-in - 老鼠
    3. 重量无菌塑料旋转袋(每个小鼠脾脏一个)到mg水平。
    4. 在处死后必须立即获得脾(注2)。在相应的感染期(例如,3-15天)后,通过颈部伸长来杀死小鼠,保持适当的伦理方案和规定(例如颈椎脱臼,CO )(Nagy等人。,2006)(注3)。
    5. 杀死后,用70%酒精彻底喷洒小鼠,并置于清洁或无菌表面上,在安全垂直层流罩内。
    6. 用小的锋利的剪刀做一个小切口到皮肤下面的腹部底部。然后打开整个腹腔,切开腹膜,暴露脾脏(位于小鼠的左上腹部)或所需的器官,按照推荐的尸检方案(Covelli,2013 http://eulep.pdn.cam.ac.uk/Necropsy_of_the_Mouse/printable.php )(注4)(图1)。


      图1.脾提取指示:A.用于尸体剖检的小鼠的位置,B.小鼠皮肤的开口,C.腹壁的开口和D.脾的提取(用白色箭头指出)。 2013年Covelli拍摄的照片。有关详情,请参阅: http://eulep.pdn.cam.ac.uk/Necropsy_of_the_Mouse/printable.php (咨询日期:09/30/2013)。

    7. 使用小镊子握住脾脏,然后与胃脾韧带一起切除hilum,以除去脾脏,并定位在预先称重的无菌塑料Whirl-Pak袋(注5)(图1)。
    8. 使用4位量表平衡重量袋内的脾脏。要获得脾脏重量,减去空的预重量塑料袋的值。

  2. 脾匀浆
    1. 加入9份含有0.1%Tween 20/g脾的PBS(稀释度1:10),假设1g脾脏的体积对应于1ml PBS(例如0.5g脾脏, 4.5ml PBS 0.1%Tween 20)。
      注意:如果在均质化过程开始时使用小体积,然后剩余体积完成到1:10稀释,均质化就会更容易。
    2. 通过用手挤压袋中的器官来精确地进行脾匀浆。
      注意:为了释放细胞内细菌,通过在含有0.1%吐温20的PBS中挤压脾组织破坏脾细胞。
    3. 样品均化后,根据预期的细菌脾定植,用1×PBS(在1.5ml eppendorf管或24孔板上)进行十倍(或所需)稀释。
      注意:小心且广泛地打开塑料袋(使用钢丝)以避免移液管污染。对于使用1×10 6 CFU的细菌的感染,范围为1-30天,平板10 -10 5 em>稀释。

  3. 电镀
    1. 建议使用两种电镀方法:
      方法A
      每个样品使用两个板(图2A),在胰蛋白胨大豆琼脂(或用于生长测试细菌的推荐培养基)板上分配三个单独的20μl的每种稀释液。然后,在孵育(5-10分钟)之前,让液滴被吸收在琼脂表面(不扩散液滴)。
      方法B
      在每个琼脂板中分配一个100微升,并用无菌Drigalski刮刀分配样品,直到接种物完全分散在琼脂表面上,每个样品使用两个板(图2B)。  
      注意:
      1. 方法A更快,并且需要更少的琼脂板,因为四个稀释液可以定位在每个板上。此外,每次稀释最多可以投放三滴,减少计数错误。如果接种101稀释液,可以检测到每克脾脏至少500个细菌。
      2. 方法B较慢并且增加了板消耗,但是当预期低细菌负荷时更加敏感。菌落计数更容易,因为每个稀释使用整个平板。如果涂覆10 1 稀释,则可检测到每g脾脏至少100个细菌。
    2. 将板在细菌菌株(例如37℃,在5%CO 2气氛下)的所需条件下培养2-3天,直到菌落形成单位(CFU)为显然。
    3. 计数各种稀释液中的CFU。只使用获得单独集落的板(和相应的稀释度)(图2A中为10-30个CFU或图2B中为30-300个CFU)。
    4. 通过将CFU乘以相应的稀释度计算脾细菌负荷,并分别通过方法A中的50或方法B中的10。  
    5. CFU方法可以通过CFU /脾脏(回收的总CFU)或者通过CFU /脾脏脾脏(回收的总CFU除以脾脏的总克数)来计算。虽然第一种方法没有考虑脾脏炎症(大小),但第二种方法校正脾肿胀。在任一情况下,总是建议在单独的图中绘制脾脏重量。


      图2.样品电镀方法

笔记

  1. 优选地,雌性小鼠(来自不同窝的雄性小鼠具有战斗的倾向),重量范围在18-22克。体重/年龄比取决于小鼠的品系。老年小鼠具有显示更多重量变化的倾向。应避免重量或年龄的差异。建议与四个小鼠的组一起工作。小鼠应在实验前处于良好的健康状况。在使用免疫抑制小鼠的情况下,推荐包括一组未感染的阴性对照小鼠以检查先前获得的细菌或酵母菌感染。
  2. 由于在组织衰变过程中微生物污染,不推荐在自然死亡或长时间后的器官中的细菌计数。
  3. 用于处死的其它方法是用诸如异氟烷(吸入> 15%)或异丙酚(静脉内> 50mg/kg)的组分的CO 2附加或过量麻醉。
  4. 避免鼠标的皮肤毛发与腹膜腔接触。
  5. 脾脏容易分离,没有大量的出血。 手术器械必须用酒精消毒并在脾脏操作之前进行发炎以避免污染。 外脏脂肪应从脾脏中清除,以避免额外的体重错误。

食谱

  1. 胰蛋白胨大豆琼脂
    将40克培养基悬浮在1升无热原型1级脱脂水中 加热频繁搅拌煮沸1分钟,使介质完全溶解
    121℃高压灭菌15分钟
  2. PBS 0.1%Tween 20
    将以下物质溶解在800ml无热原型1+级脱盐水H sub 2 O中
    8克NaCl
    0.2克KCl
    1.44g的Na 2 HPO 4
    0.24g的KH 2 PO 4 sub/
    1ml吐温20 将pH调节至7.4
    使用额外的无热原型1+级脱盐水H sub 2 O调节体积至1 L
    高压灭菌
    灭菌

致谢

我们感谢María-JesúsGrilló帮助这种技术的标准化。 该方案改编自Barquero-Calvo等人(2007)和Nagy等人(2007)和Covelli(2013)的原始发表的论文。 这项工作由FIDA-2006,FS-CONARE(UNA/UCR),MICIT/CONICIT(FI-487-09),8-N-2005和B/3456-1(NeTropica),B/3456- 2(IFS),AGL2004-01162/GAN(西班牙),CNRS和INSERM(法国)和MASTERSWITCH项目(欧洲共同体)。

参考文献

  1. Barquero-Calvo,E.,Chaves-Olarte,E.,Weiss,D.S.,Guzman-Verri,C.,Chacon-Diaz,C.,Rucavado,A.,Moriyon,I.and Moreno,E。 Brucella abortus 使用隐形策略来避免先天免疫系统的激活 在感染发作期间。 PLoS One 2(7):e631
  2. Covelli,V.(2013)。 鼠标尸检指南。
    http://eulep.pdn.cam.ac.uk/Necropsy_of_the_Mouse/printable.php
  3. Nagy,A.,Gertsenstein,M.,Vintersten,K。和Behringer,R。(2006)。 通过颈椎脱位快速和人道地牺牲小鼠。 CSH Protoc 2006(1)。    
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Barquero-Calvo, E., Chacón-Díaz, C., Chaves-Olarte, E. and Moreno, E. (2013). Bacterial Counts in Spleen. Bio-protocol 3(21): e954. DOI: 10.21769/BioProtoc.954.
提问与回复

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当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。