Microbial DNA - Purify the easy way!

Challenges of nucleic acid purification from bacteria or yeasts 

Bacteria and yeast are two of the major groups classified as microogranisms. Bacteria are prokaryotic single-celled organisms without membrane-bound organelles or nucleus. They can be divided into gram-positive or gram-negative bacteria, based on their membrane compositions. Yeast, on the other hand are eukaryotic single celled organisms with cell organelles and a nucleus. One of their membrane building blocks is chitin. 

To obtain the best possible results for your sample, please read below about challenges and our suggested solutions to purify nucleic acids from bacteria and yeast used in research labs or as starter culture for industrial processes.

Our solutions for your challenges

Read below about our selected lysis and nucleic acid purification for your samples. Please also find some more insights about the cell wall structure of bacteria and yeast which plays and essential role for lysis and nucleic acid purification. 

• Important differences in cell wall structure
• DNA purification solutions from bacteria and yeast
• Lysis solutions for bacteria and yeast
• Solutions for starter culture microorganism used in industrial processes

Important differences in cell wall structure

The cell wall protects the cell, provides structure and support. Cell walls of all microorganisms do differ in their compostion. For yeast and bacteria the main difference in membrane compostion is the presence of chitin in yeast cell walls, whereas in bacteria the cell wall is made of peptidoglycan. Understanding the cell wall structure in detail will also help to choose the best lysis option for your sample. Read more about the structural differences of bacterial and yeast cell walls below.

Bacteria

Using the Gram-staining, bacteria can be categorized into two groups, due to the peptidoglycane structure in their cell walls. The membrane structure is also important when choosing a lysis method. Click here to read more about lysis suggestions for gram-negative and gram-positive bacteria.

Gram-positive

Gram-positive bacteria show a complex membrane structure which includes peptidoglycans, polysaccharides, teichoic acids and proteins. With 40 to 80 layers of peptidoglycan, the gram-positive cell wall is relatively thick compared to cell walls of gram-negative bacteria.

Classification of gram-positive bacteria

Gram-positive bacteria are divided into the following groups based on their shape

1. Cocci, such as Staphylococcus or Streptococcus

2. Bacilli, which can be further divided into spore-forming, such as Bacillus and Clostridia or non-spore forming, such as Listeria and Corynebacterium

Gram negative

Gram-negative bacteria appear red after the gram staining. In addition to their structural petidoglycan membrane composition, which makes them less susceptible for certain antibiotics such as penicillin, they also are protected via an outer capsule. This outer capsule allows further protection against antibiotics and can release endotoxins when disrupted.

Classification of gram-negative bacteria

Gram-negative bacteria can be divided into

1. Diplococci, that are either maltose utilizing such as Neisseria gonorrhoeae or non-maltose utilizing like Neisseria meningitidis

2. Coccobacilli, such as H. influenzae or B. pertussis

3. Bacilli which are either lacose fermenting, such as E. coli or non-lactose fermenting but H2S-producing such as S. proteus 

4. Comma-shaped oxidase positive, such as V.cholerae or H.pylori

Yeast

The cell wall structure of fungi or yeast has four characteristics: glucans, chitin, glycoproteins and melanin. Differences in the composition of the four allows the characterisation of different species.

Glucans

• Glucan represents 50–60% of their cell wall
• The most abundant glucans are 1,3-linked glucose units (65–90%)
• Cell wall components are covalently linked to the 1,3-glucan units

Chitin

• The content of chitin within the fungal cell wall varies due to their morphological phase (i.e. budding)
• Chitin make up to 1-2% of the cell wall
• Filamentous fungi show up to 12% of chitin within their cellwall

Lysis solutions for bacteria and yeast

Lysis via lytic enzymes

Enzymatic lysis suggestions for bacteria

Proteinase K

Proteinase K is available in two different versions

• Lyophilized Proteinas K
• Liquid Proteinase K

Enzymatic lysis suggestions for yeast

• Lyticase or zymolase
  

Mechanical lysis solutions

Mechanical lysis solutions for bacteria

MN Bead Tubes

MN Bead Tubes Type B, 40-400 µm glass beads

The MN Bead Tubes Type B are available in the following formats:

• MN Bead Tubes Type B 2 mL
• MN 96 Bead Plate Type B
• MN Bead Tubes Bulk, B1 and B2

Mechanical lysis solutions for yeast

MN Bead Tubes

MN Bead Tubes Type C, 1-3mm corundum beads

The MN Bead Tubes are available in the following formats

• MN Bead Tubes Type C, 2 mL
• MN Bead Tubes Type C Bulk

Solutions for starter culture microorganisms used in industrial processes

For the fermentation of food, various species of bacteria or yeast are used. For the production of meat, cheese or dairy products, mostly bacteria are used. Yeasts are used for the pruduction of bread, beer or wine.

For each of the mentioned products the bacteria or yeasts, that are used within a starter culture, are carefully selected and characterized.

Starter Cultures - Bacteria

• Lactococcus lactis, used for dairy products or cheese
• Streptococcus thermophilus, used for cheese or yoghurt
• Pediococcus halophilus,used for soy sauce
• Pediococcus acidilactici, used for sausage or fermentation of vegetables
• Oenococcus oeni, used for wine

Starter Cultures - Yeast

• Saccharomyces cerevisisiae, bread production, ale beers or wine
• Saccharomyces calsbergensis, lager beers
• Kluyveromyces spp., production of kefir
• Candida spp., production of kefir
• Brettanomyces bruxellensis, production of beer
• Brettanomyces spec., production of cider or apple wines
  

Solutions for starter cultures

NucleoMag DNA Bacteria

• suitable for the use of starter cultures
• isolation from diverse cultures
• fully automatable

The NucleoMag DNA Bacteria kit can be used manually together with the NucleoMag Sep Mini for single preps or with the NucleoMag SEP for 96 samples. Automation is possible on the common automation platforms like Eppendorf, Hamilton, Tecan. Please contact our Technical Support for more information.

If a mechanical lysis is needed the MN Bead Tubes are available as single tube version or as MN 96 Bead Plate. For more information on the MN Bead Tubes click here.

DNA purification solutions from bacteria and yeast

NucleoSpin Microbial DNA

• Suitable for a large variety of starting materials
• MN Bead Tubes Type B for efficient lysis included – compatible with many disruption devices
• Fast and easy procedure – Liquid Proteinase K included, no additional enzyme required

NucleoSpin Microbial DNA procedure

NucleoSpin Microbial DNA is designed for rapid purification of highly pure genomic DNA from microorganisms (gram-negative and gram-positive bacteria, yeast, and fungi).

The NucleoSpin Microbial DNA kit utilizes MN Bead Tubes Type B (glass beads; included in the kit) in combination with Liquid Proteinase K, and a common disruption device e.g., swing mill, in order to lyse different sample materials with one procedure. The procedure is fast, simple and reliably high DNA yields from a wide range of microbial samples can be obtained. Alternatively MN Bead Tubes can be ordered separately. 

Appropriate DNA binding conditions to the NucleoSpin Microbial DNA Columns are achieved by addition of large amounts of chaotropic salts (Binding Buffer MG) to the lysate. Contaminants are removed by two efficient washing steps. Afterwards, DNA is eluted with a slight alkaline buffer and is ready-to-use for subsequent reactions.

Efficient DNA recovery from different microorganisms

DNA was isolated with the NucleoSpin Microbial DNA kit and MN Bead Tube Type B (included in the kit) or MN Bead Tube Type C (see ordering information). 100 ng DNA per prep were analyzed by agarose gel electrophoresis, showing high molecular DNA, without RNA contamination or DNA degradation. 

1. Escherichia coli, MN Bead Tube Type B 

2. Vibrio fischeri, MN Bead Tube Type B 

3. Bacillus subtilis, MN Bead Tube Type B 

4. Corynebacterium glutamicum, MN Bead Tube Type B 

5. Saccharomyces cerevisiae, MN Bead Tube Type C

Comparison of preparation times

The prep time of NucleoSpin Microbial DNA kit was compared with competitor products by processing Bacillus subtilis, Escherichia coli, and Saccharomyces cerevisiae. The NucleoSpin Microbial DNA kit shows shorter prep times in all three cases.

Various applications are possible in combination with the MN Bead Tubes

DNA was successfully isolated from the above mentioned microorganisms by the NucleoSpin Microbial DNA kit. The MN Bead Tubes were tested for the mentioned sample materials on a Retsch Swingmill MM300, operating at highest frequency (30 Hertz). The time and frequency might have to be optimized for other disruption devices, as well as other sample materials.

NucleoSpin DNA Yeast

• Optimized lysis with MN Bead Tubes Type C
• Up to 100 mg of sample input
• High genomic DNA yields

For DNA from yeast: Don’t lyse hard, lyse smart! 

Protocols for isolation of DNA from yeast typically include either a chemical treatment to weaken the cell walls, or a mechanical lysis with glass beads. The former requires additional hands-on time, the latter often is inefficient, leading to prolonged lysis times and low DNA yields. The NucleoSpin DNA Yeast kit contains MN Bead Tubes Type C with corundum beads for most efficient lysis. Using corundum beads improves the lysis, resulting in a drastically increased DNA yield. The kit delivers superior yields for various popular yeast species, including Saccharomyces cerevisiae, Schizosachharomyces pombe and Pichia pastoris.

High DNA yields and efficient sample disruption from up to 100 mg sample

NucleoMag DNA Bacteria

• Environmentally sustainable buffer chemistry – no chaotropic salts

• Combine with MN Bead Tubes for single sample processing or MN 96 Bead Plates for high-throughput sample disruption

• Liquid Proteinase K and Liquid RNase A for easy handling

NucleoMag DNA Bacteria

The NucleoMag DNA Bacteria kit enables high-throughput, automation-friendly isolation of DNA from diverse microbial samples. The kit is optimal for cultured Gram-positive and Gram-negative bacteria, yeast, and spores. NucleoMag DNA Bacteria utilizes a powerful, yet environmentally friendly buffer chemistry, free of chaotropic salts, as well as any dangerous goods (patent pending). The kit can be combined with MN Bead Tubes or MN 96 Bead Plates for mechanical disruption.

Competitive detection of microbial DNA

DNA was isolated from Gram-positive and Gram-negative bacteria, as well as yeast using the NucleoMag DNA Bacteria kit (MN, blue bars) in comparison to competitor kits Q and T (grey bars). All procedures were performed according to manufacturer‘s recommendations. In comparison to competitors Q (figure A) and T (figure B) the PCR results show significantly earlier amplification (lower CT values), demonstrating superior extraction of microbial DNA. The qPCR was performed for 16S rRNA and 18S rRNA for bacteria and yeast, respectively, using the Maxima SYBR Green kit from Thermo Scientific on Applied Biosystems 7500 Real-Time PCR System.

Efficient DNA extraction with both bead plate and single tube homogenization

DNA was isolated from various Gram-positive and Gram-negative bacteria using the NucleoMag DNA Bacteria kit in combination with different solutions for sample homogenization. DNA yields were determined by UV spectrometry. The samples were homogenized by using either racks of prefilled tube strips (MN 96 Bead Plates), or single bead tubes (MN Bead Tubes). The results for samples homogenized with MN 96 Bead Plates (dark blue bars) were comparable to the results obtained with homogenization in the MN Bead Tubes (light blue bars).

Reliable DNA integrity with bead plate or single bead tube homogenization

DNA was isolated from Gram-positive and Gram-negative bacteria using the NucleoMag DNA Bacteria kit. The samples were simultaneously homogenized by using either a rack of prefilled tube strips (BP = MN 96 Bead Plate) or single bead tubes (BT = MN Bead Tubes). Both homogenization systems enable a reliable DNA extraction in combination with the NucleoMag DNA Bacteria kit.

Overview of successfully tested sample materials

The NucleoMag DNA Bacteria kit has been evaluated with several different sample types and species. Successful DNA isolation was verified via agarose gel electrophoresis or qPCR.